Oligosaccharides and oligosaccharide-protein conjugates derived from Clostridium difficile polysaccharide PS-I, methods of synthesis and uses thereof, in particular as vaccines and diagnostic tools

ABSTRACT

The invention relates to a synthetic oligosaccharide representing part of the repeating unit of the  Clostridium difficile  glycopolymer PS-I and having the sequence of the pentasaccharide a-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[a-L-Rhap-(1→3]-a-D-Glcp-(1→2)-a-D-Glcp or a synthetic fragment or derivative thereof. Preferably, the claimed synthetic oligosaccharide bears at least one linker L for conjugation to a carrier protein or for immobilization on a surface. Further aspects of the invention relate to advantageous methods for synthesizing said synthetic oligosaccharide and oligosaccharide-protein conjugate as well as to uses thereof, in particular as vaccines and diagnostic tools.

BACKGROUND

Clostridium difficile is a Gram-positive, spore forming anaerobicbacterium that colonizes the intestinal tract of humans thus leading toC. difficile infections (CDI). CDI has become the most commonlydiagnosed cause of hospital-acquired diarrhea, particularly in the riskgroups including elderly and immunodeficient patients as well as thosereceiving antibiotic treatment. A steep rise in CDI incidents over thepast decade is attributed to the emergence of the hypervirulent, and nowpredominant strain ribotype 27, causing epidemic outbreaks withincreased morbidity, mortality and high relapse rates. The costs totreat patients have greatly increased, particularly in the case ofrecurring CDI. Preventive methods, such as vaccination of risk groups,may be useful and cost-efficient means to avoid future infections.Although vaccination against C. difficile should be economicallyfeasible (B. Y. Lee et al., Vaccine, 2010, 28, 5245) a vaccine has notyet been developed.

Carbohydrates exposed on the cell-surface of pathogens are oftenimmunogenic and constitute potential candidates for vaccine development.When covalently connected to carrier proteins, carbohydrate antigenvaccines can elicit a long lasting T-cell dependent protection (C.Snapper and J. Mond, J. Immunol., 1996, 157, 2229). Several vaccinescontaining carbohydrates, isolated from biological sources, are inroutine use (G. Ada and D. Isaacs, Clin. Microbiol. Infect., 2003, 9,79). Vaccines based on synthetic carbohydrate antigens against bacteria,viruses, parasites and cancer are currently in preclinical and clinicaldevelopment (a) R. D. Astronomo and D. R. Burton, Nature Rev., 2010, 9,308; b) M.-L. Hecht, P. Stallforth, D. V. Silva, A. Adibekian and P. H.Seeberger, Curr. Opin. Chem. Biol., 2009, 13, 354).

The chemical structure of two C. difficile cell-surface polysaccharides,PS-I and PS-II has been elucidated recently (J. Ganeshapillai et al.,Carbohydr. Res., 2008, 343, 703; WO 2009/033268 A1). Initial focus hasbeen turned towards the PS-II hexasaccharide antigen that is believed tobe common to several C. difficile strains (a) E. Danieli et al., Org.Lett., 2010, 13, 378; b) M. Oberli, M.-L. Hecht, P. Bindschädler, A.Adibekian, T. Adam and P. H. Seeberger, Chem. Biol., 2011, 18, 580). Thesynthetic PS-II hapten is immunogenic when conjugated to a carrierprotein and antibodies found in the stool of C. difficile patients bindto the synthetic PS-II hexasaccharide (Oberli et al., ibid.). Thepentasaccharide phosphate repeating unit PS-I was reported as[→4)-α-Rhap-(1→3)-β-Glcp-(1→4)-[α-Rhap-(1→3)]-α-Glcp-(1→2)-α-Glcp-(1→P]and it is suggested to be specific for the strain ribotype 27.

In conclusion, the pathogen C. difficile represents a major risk forpatients and causes significant costs to health care systems.Unfortunately, however, currently no licensed vaccine against C.difficile is available.

Thus, a main object of the present invention is to provide novel andeffective means to prevent and/or to treat C. difficile associateddiseases, in particular related to the hypervirulent strain ribotype027. A further object is to provide novel and effective means to detectC. difficile in a sample and/or a C. difficile infection in a subject. Afurther object is to provide novel and effective means to identify acertain strain of C. difficile in a sample and/or a C. difficileinfected subject. A further object is to provide novel and effectivestandards for immunoassays for the detection of C. difficile.

The present inventors succeeded in the first total synthesis of apentasaccharide derived from the repeating unit of the C. difficilepolysaccharide PS-I, and its conjugation to the diphtheria toxoidCrm₁₉₇.

Consequently, the above main object of the invention is achieved byproviding the synthetic oligosaccharide, in particular pentasaccharide,the oligosaccharide-protein conjugate and the composition according tothe invention. Further objects are achieved by providing the antibody,the methods of detection and identification, and the methods ofsynthesis according to the invention.

DESCRIPTION OF THE INVENTION

The present invention provides an oligosaccharide, in particularsynthetic oligosaccharide, derived from the repeating unit of theClostridium difficile glycopolymer PS-I and an oligosaccharide-proteinconjugate comprising said oligosaccharide coupled to a protein carrier.

More specifically, the oligosaccharide is the pentasaccharide having thesequenceα-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3]-α-D-Glcp-(1→2)-α-D-Glcp ora (synthetic) fragment or derivative thereof.

The term “derivative” as used herein means generally any structurallyrelated molecule having the same scaffold as the basic molecule butwhich is modified by the addition, deletion or substitution of one ormore functional groups. For example, the “oligosaccharide derivative” asused herein may be obtained by replacement of one or more of thehydroxyl groups by other functional groups or atoms or by introducingadditional substituents such as linker groups.

The term “fragment” as used herein includes tetra-, tri-, di- andmonosaccharides which are constituting units of the pentasaccharidehaving the sequenceα-L-Rhap-(1→3)-α-D-Glcp-(1→4)-[α-L-Rhap-(1→3]-α-D-Glcp-(1→2)-α-D-Glcpfrom above or from a derivative thereof, in particular a derivativecomprising one or more linker group(s).

Preferably, the oligosaccharide bears at least one linker L forconjugation to a carrier protein or for immobilization on a surface.

The linker or spacer group L may be any moiety that enables to couplethe oligosaccharide to a carrier molecule or to the surface of amicroarray. A large variety of such linker groups are known in the artand a suitable linker group can be selected in dependence from therespective carrier molecule or surface group. For example, L may be analiphatic or aromatic residue, e.g. an alkyl(en) group or phenyl(en)group, comprising a reactive functional group, such as an amino group,preferably a primary amino group, (activated) carboxy group, aldehyde,azide, alkenyl or alkinyl group. In specific embodiments L may comprisea polyether or polyester chain. In particular, L is selected from thegroup comprising primary alkylamines, alkyl or aralkyl residues with aterminal aldehyde, azide, alkine or alkene group or (activated) carboxygroup, and alkylaryl and aryl residues, e.g. phenyl residues, comprisinga reactive amine, aldehyde or azide group, or (activated) carboxy group.

In a specific embodiment of the invention, L is (CH₂)_(n)NH₂, with nbeing an integer from 2 to 50, preferably 3 to 20 or 3 to 10, such as 4to 8.

The carrier may be any carrier molecule known in the art, in particularin the field of vaccine development, e.g. as disclosed in Hecht et al.,Curr. Opin. Chem. Biol. 13, 354-359. (2009). More specifically thecarrier is a protein carrier selected from the group comprisingdiphtheria toxoid CRM₁₉₇, tetanus toxoid (TT), outer membrane protein(OMP), bovine serum albumin, (BSA), keyhole limpet hemocyanine (KLH),diphtheria toxoid (DT), cholera toxoid (CT), recombinant Pseudomonasaeruginosa exotoxin A (rEPA), Clostridium difficile toxin A (TcdA),Clostridium difficile toxin B (TcdB).

The synthetic pentasaccharide derived from the repeating unit of C.difficile PS-I will induce an immunogenic and antigenic response inmice, livestock and human patients.

Consequently, an aspect of the present invention relates to a vaccineagainst the pathogen Clostridium difficile comprising at least one ofthe group consisting of the synthetic oligosaccharide, theoligosaccharide-protein conjugate, or a conjugate of the oligosaccharideaccording to the invention or derivative thereof with a non-proteincarrier molecule.

The oligosaccharide-protein conjugate or the oligosaccharide, inparticular the pentasaccharide, of the invention may be advantageouslyused for preparing a pharmaceutical composition for the treatment orprevention of a disease caused by a pathogenic strain of Clostridiumdifficile.

In a related aspect they may be used in a method for the treatment orprevention of a disease caused by the pathogen Clostridium difficile.

In a further related aspect they may be used as diagnostic tools fordetecting Clostridium difficile or identifying a certain strain ofClostridium difficile in a sample and/or a Clostridium difficileinfection in a subject. Such a method may be, e.g. a diagnostic methodfor Clostridium difficile infection comprising the use of the syntheticoligosaccharide of the invention or a mixture thereof. They may forexample be used as effective standards for immunoassays for thedetection of C. difficile.

A further aspect of the invention relates to an antibody havingspecificity for an immunogenic determinant derived from or comprisingthe repeating unit of the Clostridium difficile glycopolymer PS-I. Morespecifically, the immunogenic determinant comprises or consists of thepentasaccharide of the invention.

In a specific embodiment, said antibody has been raised against aoligosaccharide-protein conjugate wherein the oligosaccharide is thepentasaccharide 1 or a derivative thereof and the protein carrier isdiphtheria toxoid CRM₁₉₇.

The antibody may be a polyclonal or monoclonal antibody and monoclonalantibodies can be readily prepared by standard methods of the art (e.g.Köhler and Milstein (1975), Nature, 495-497).

The present invention also provides very favourable and efficientmethods for synthesizing the pentasaccharide and pentasaccharide-proteinconjugates selectively and in high yields.

These methods involve the use of one or more of molecules 2, 2′, 3, 4,5, 20, 21, 22, 23, 24, 27, 29, 30, 30′, 31, 32, 32′, 33, 33′, 34′ asshown or defined below as intermediates or building blocks for preparingthe pentasaccharideα-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcpor of a derivative thereof.

A first preferred method (method A) for synthesizing the pentasaccharide1 shown in Scheme 1 below

comprises assembling the monosaccharide building blocks 2 and 3 or 4shown in Scheme 1 to yield the corresponding disaccharide 21 of Scheme5, reacting the disaccharide 21 with building block 4 to form thetrisaccharide 23 of Scheme 5, subjecting the trisaccharide 23 to abis-glycosylation reaction with 2 molecules of building block 5 shown inScheme 1 to yield the fully protected pentasaccharide 24 in Scheme 5 andfinally, after deprotection, to yield pentasaccharide 1.

This method can be generalized for preparing other pentasaccharideshaving the sequenceα-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcp-Laccording to the invention wherein the specific amino linker of compound1 is replaced by any linker L, in particular any linker L as definedherein. This linker may also be present on a position (sugar moiety)different from the specific position (sugar moiety) indicated above. Thegeneralized method comprises assembling a monosaccharide building block2′, wherein the specific protected amino linker of building block 2 isreplaced by a protected or unprotected linker L, in particular a linkerL as defined herein, and building blocks 3 or 4 shown in Scheme 1 toyield the corresponding disaccharide 21′, reacting the disaccharide 21′with building block 4 to form the trisaccharide 23′, subjecting thetrisaccharide 23′ to a bis-glycosylation reaction with 2 molecules ofbuilding block 5 shown in Scheme 1 to yield the fully protectedpentasaccharide 24′ in Scheme 5 and finally, after deprotection, toyield pentasaccharide 1′, wherein the specific amino linker ofpentasaccharide 1 is replaced by a different linker L, in particular alinker L as defined herein.

The method for preparing the oligosaccharide-protein conjugate of thepresent invention typically comprises coupling the oligosaccharide ofthe invention bearing a linker or spacer group L, in particular whereinL is (CH₂)_(n)NH₂, with n being an integer from 2 to 50, preferably from3 to 20, with a protein carrier.

More specifically, said method comprises providing a pentasaccharidehaving the sequenceα-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3]-α-D-Glcp-(1→2)-α-D-Glcp-Lbearing a linker L=(CH₂)_(n)NH₂, with n being an integer from 2 to 50,preferably from 3 to 20, and reacting the unique terminal amine of thelinker L with one of the two NHS-activated esters of Di(N-succinimidyl)adipate to form an amide and subsequent coupling of the activated amidemoiety to the protein carrier. The protein carrier may be any carrierdisclosed above and in one specific embodiment the protein carrier isCRM₁₉₇.

In the following, the methods of synthesis according to the inventionare outlined in more detail with respect to preferred embodiments butare not limited thereto.

General Oligosaccharide Synthesis

The present inventors developed very effective methods for synthesizinga pentasaccharide having the sequenceα-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcp-Lthat comprises the PS-I repeating unit but differs from the naturalpentasaccharide by the linker L. In a preferred embodiment, theoligosaccharide was designed to carry a primary amine at the reducingterminus via a linker to facilitate conjugation to a protein carrier andattachment to microarrays or other surfaces. Based on the retrosyntheticanalysis (Scheme 1), the pentasaccharide 1—wherein the linker comprisesthe (CH₂)₅NH₂ group—can be assembled from the monosaccharide buildingblocks 2 and 3 or 4, and the monosaccharide building block 5 and theseassembling steps are outlined in more detail below.

However, it is to be understood that analogous assembling steps can beperformed using an analogous building block 2′ differing from buildingblock 2 only by the presence of a different linker, in particular suchas defined herein, resulting in an analogous pentasaccharide 1′.

The 1,2-cis glycosidic linkages of the glucose residues A and B wereinstalled early in the synthesis by employing the non-participatingprotecting groups 2-naphthylmethyl (NAP) and benzyl in 2-positions. Thetemporary protecting groups Lev and Fmoc present in the glucose buildingblocks B and C were chosen for their compatibility with automated solidphase synthesis (K. R. Love and P. H. Seeberger, Angew. Chem. Int. Ed.,2004, 43, 602). Both Rha residues D and D′ were installed in a singlebisglycosylation reaction.

Following placement of the NAP-protection in thioglycoside 6 (S. J.Danishefsky, S. Hu, P. F. Cirillo, M. Eckhardt and P. H. Seeberger,Chem. Eur. J., 1997, 3, 1617) the terminal linker carrying a latentamine was introduced by union of thioglucoside 7 and the linker prior tosubsequent DDQ-mediated cleavage of the C-2 napthyl ether in order toproduce glucose building block 2 (Scheme 2) a) J.-G. Delcros, S. Tomasi,S. Carrington, B. Martin, J. Renault, I. S. Blagbrough and P. Uriac, J.Med. Chem., 2002, 45, 5098; b) J. Xia, S. A. Abbas, R. D. Locke, C. F.Piskorz, J. L. Alderfer and K. L. Matta, Tetrahedron Lett., 2000, 41,169)

The synthesis of thioglucoside 11 that served as common precursor forbuilding blocks 3 and 4 commenced from β-d-glucose pentaacetate 8(Scheme 3). Use of the nontoxic and odorless2-methyl-5-tert-butyl-thiophenol group ensured exclusive formation ofβ-anomer of thioglucoside 9 (M. Collot, J. Savreux and J.-M. Mallet,Tetrahedron, 2008, 64, 1523). The acetyl groups were removed and the 4-and 6-hydroxyl groups of the resulting tetraol were regioselectivelyprotected as a 4,6-O-benzylidene acetal (J. S. S. Rountree and P. V.Murphy, Org. Lett., 2009, 11, 871) to afford diol 10. Regioselectiveplacement of a TBS-ether protecting group at the 3-OH gave thioglycoside11 (K. C. Nicolaou, N. Winssinger, J. Pastor and F. DeRoose, J. Am.Chem. Soc., 1997, 119, 449).

Synthesis of building block 3 began with the installation of thenon-participating benzyl group at the 2-position of 12 to favor theformation of the α-glycosidic linkage between monosaccharides A and Bfragments. Subsequent placement of the 3-O-Fmoc-protection furnishedcompound 13. Finally, the regioselective opening of the4,6-O-benzylidene acetal with TES-TfOH and protection of the free4-hydroxyl gave orthogonally protected building block 3. Preparation ofdifferentially protected glucosyl phosphate 4 from 11 followed a similarroute. In anticipation of the formation of a 1,2-trans linkage betweenthe B and C saccharide fragments, a participating benzoyl group wasinstalled at the 2-position of 15. During TBAF-mediated desilylation of15, careful control of the TBAF:AcOH ratio was essential to preventbenzoyl-migration from the C2- to C3-positions. Fmoc-protectedthioglycoside 17 was further converted to glycosyl phosphate 4.

Synthesis of the rhamnosyl building block 5 to provide the D fragmentcommenced with the bis-benzoylation of 4-methoxyphenyl glycoside 18(Scheme 3) (D. B. Werz, A. Adibekian and P. H. Seeberger, Eur. J. Org.Chem., 2007, 12, 1976). CAN-mediated removal of the anomericp-methoxyphenyl group yielded the free lactol that was immediatelyconverted into rhamnosyl N-phenyl trifluoroacetimidate 5 (B. Yu and H.Tao, Tetrahedron Lett., 2001, 42, 2405).

The assembly of the pentasaccharide target was achieved in seven linearsteps by combining the monosaccharide building blocks in sequence(Scheme 5).

Installation of the α-glycosidic linkage was the result of the union ofglycosylating agent 3 and nucleophile 2. Disaccharide 20 was obtained ingood yield and stereoselectivity when NIS and TfOH in Et₂O was employedas promoter system. Selective cleavage of the levulinic ester withhydrazine hydrate in pyridine/AcOH, did not compromise the integrity ofthe Fmoc-group but cleanly produced disaccharide acceptor 21.Thioglucoside building block 17, a very storage-stable monomer unit hadbeen intended for the installation of the next glycosidic linkage toform trisaccharide 22. Upon a variety of conditions only traces of thedesired product 22 were isolated. As a first means to remedy thesituation, replacement of the anomeric leaving group was executed.Glycosyl phosphate 4 was activated by TMSOTf to promote theglycosylation of 21 and afforded 22, although purification was achievedonly following Fmoc cleavage to yield 23. Conversion of diol 23 to fullyprotected pentasaccharide 24 was achieved by bis-glycosylation usingrhamnosyl-imidate 5 in the presence of TMSOTf. Final deprotection ofcompound 24 required two transformations: saponification of the benzoateesters and catalytic hydrogenation of the aromatic groups gavepentasaccharide 1. Careful comparison of the spectroscopic data forsynthetic pentasaccharide 1 and NMR spectra of native PS-I revealedexcellent agreement.

In summary, the first synthesis of the C. difficile cell-surface PS-Ipentasaccharide 1 was achieved employing a linear strategy that servesto scout reaction conditions for automated solid phase synthesis and toidentify robust and efficient monosaccharide building blocks. Four suchbuilding blocks 2-5 were prepared. Glycosyl phosphate 4 proved asignificantly better building block than identically protectedthioglycoside 3.

The present inventors also developed an alternative route of synthesisbased on a similar strategy as outlined above which is even moreefficient and results in greatly improved yields of the PS-Ipentasaccharide product.

The innovation of this improved synthesis relies on the use of theprotecting group para-bromobenzyl (PBB) [Plante et al., J. Am. Chem Soc.122:7148-7149, 2000; Liu et al., Chem. Commun. 1708-2709; 2004].Building block 27, modified with PBB at C-3 was obtained in three stepsfrom intermediate 12 described above (Scheme 3). PBB-containing 27 wasused for the following pentasaccharide synthesis rather thanFmoc-containing 3 used in the method outlined above.

A further improvement of the previous synthesis was achieved byreplacing the acid-labile building block 4 with more stable 29. The4,6-O-benzylideneacetal ring of previously reported intermediate 15 wasopened selectively, followed by

Assembly of the pentasaccharide took place similarly as described abovefor method A; changes were made in the deprotection steps d), e) and f)(Scheme 8) due to the modified protective group pattern.

Synthesis of the pentasaccharide 1 according to method B preferablycomprises assembling the monosaccharide building blocks 2 and 27 shownin Scheme 8 to yield the corresponding disaccharide 30 of scheme 8,reacting the disaccharide 30 with building block 4 or 29 to form theprotected trisaccharide 32 of scheme 8, deprotecting the trisaccharide32 to obtain trisaccharide 33 and subjecting trisaccharide 33 to abis-glycosylation reaction with 2 molecules of building block 5 shown inScheme 8 to yield the fully protected pentasaccharide 34 in Scheme 8 andfinally, after deprotection, to yield pentasaccharide 1.

Formation of the Glc(1→4)Glc linkage (Scheme 8, step c) proceeded in 92%yield, a huge improvement compared to 38% in method A.

This method can be generalized for preparing other pentasaccharideshaving the sequenceα-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcp-Laccording to the invention wherein the specific amino linker of compound1 is replaced by any linker L, in particular any linker L as definedherein. This linker may also be present on a position (sugar moiety)different from the specific position (sugar moiety) indicated above. Thegeneralized method comprises assembling a monosaccharide building block2′, wherein the specific protected amino linker of building block 2 isreplaced by a protected or unprotected linker L, in particular a linkerL as defined herein, and building block 27 shown in Scheme 8 to yieldthe corresponding disaccharide 30′, reacting the disaccharide 30′ withbuilding block 4 or 29 to form the corresponding protected trisaccharide32′, deprotecting the trisaccharide 32′ to obtain trisaccaride 33′,subjecting the trisaccharide 33′ to a bis-glycosylation reaction with 2molecules of building block 5 shown in Scheme 1 to yield the fullyprotected pentasaccharide 34′ and finally, after deprotection, to yieldpentasaccharide 1′, wherein the specific amino linker of pentasaccharide1 is replaced by a different linker L, in particular as defined herein.

Synthesis of PS-I Substructures

A comprehensive set of PS-I substructures 35-39 (Scheme 9) carrying anamino-linker was synthesized. The pentasaccharide repeating unit 1 isbuilt up from glucose residues A, B and C and terminal rhamnoses D andD′. Disaccharide 35 contains A and B, trisaccharide 36 A, B and C. Thesequence BCD′ is covered by trisaccharide 37. Disaccharide 38 coversboth the BD and CD′ sequence. Rhamnose substructure 39 represents D andD′.

Oligoglucose disaccharide 35 (Scheme 10) and trisaccharide 36 (Scheme11) were obtained by catalytic hydrogenation of protected disaccharide31 and trisaccharide 33.

Oligosaccharides 38 (Scheme 12) and 37 (Scheme 13) containing a terminalrhamnose residue were synthesized relying on disaccharide 41 which inits turn was obtained by union of 40 and 5.

Rhamnoside 39 (Scheme 14) bearing an anomeric linker was attained bycombining 5 and 5-aminopentanol.

Microarray-chips containing 1 and the substructures 36-39 were prepared.This set of oligosaccharides substructures covalently linked to asurface was used to identify binding epitopes of anti PS-Ipentasaccharide antibodies raised in mice (FIG. 6).

The pentasaccharide 1 or 1′ obtained as outlined above or a fragment orderivative thereof can be coupled to a carrier protein by a variety ofknown methods.

A particular advantageous and preferred method uses the approach shownin scheme 15 below. For this the unique, terminal amine of 1 was firstreacted with one of the two NHS-activated esters of Di(N-succinimidyl)adipate to form an amide. The coupling of the activated pentasaccharideto CRM₁₉₇ proceeded in phosphate buffer (any other usual bufferproviding the desired pH is also suitable) and in one experimentresulted in a load that averaged 3.6 pentasaccharide units per protein,as determined by MALDI-TOF mass spectrometry. However, otherpentasaccharide loads (such as e.g. about 9.6 units per carriermolecule) are also possible by varying the reaction conditions (compareExample 3).

Microarray Chips

Oligosaccharides, in particular pentasaccharide 1 and substructures 35through 39, were immobilized on the surface of NHS-activated glassslides via their terminal primary amine group of the linker moiety.These microarrays were used to detect and quantifyoligosaccharide-specific antibodies.

Polyclonal and Monoclonal Antibodies

Monoclonal antibodies (mABs) were generated using the standard method byKöhler and Milstein, 1975. These showed specificity for pentasaccharide1.

The invention is further illustrating by the following non-limitingExamples and Figures.

FIGURES

FIG. 1. Glycoconjugate 1 composed of hapten 1 (pentasaccharide 1) andprotein CRM₁₉₇

FIG. 2. Characterization of glycoconjugate 1a; a) SDS-PAGE; b)MALDI-TOF; c) HPLC

FIG. 3. Conjugate reaction resulting in glycoconjugate 1b

FIG. 4 a. SDS-PAGE analysis of CRM₁₉₇ glycoconjugate 1b

FIG. 4 b. MALDI-TOF MS analysis of CRM₁₉₇ glycoconjugate 1b

FIG. 5. Microarray design

FIG. 6. Microarray analysis of immune response against glycoconjugate.Dilutions of pooled sera in PBS are indicated under the microarrayimages.

FIG. 7. Antibody titers against the PS-I pentasaccharide (left), CRM₁₉₇(center), and spacer moiety (right), as determined by microarrayanalysis

FIG. 8. Isotype analysis of the immune response against PS-Ipentasaccharide

FIG. 9. Microarray design including PS-I pentasaccharide 1 andsubstructures thereof, 35 through 39

FIG. 10. Immune response against PS-I substructures of mice immunizedwith PS-I glycoconjugate without adjuvant

FIG. 11. Immune response against PS-I substructures of mice immunizedwith PS-I glycoconjugate and Freund's adjuvant

FIG. 12. Immune response against PS-I substructures of mice immunizedwith PS-I glycoconjugate and Alum adjuvant

FIG. 13. Isotype analysis of monoclonal antibodies and theirreactivities against PS-I substructures

EXAMPLE 1 Preparation and Characterization of a Pentasaccharide Based onthe Repeating Unit of C. difficile Polysaccharide PS-I

The pentasaccharide was designed to provide, by means of a linker group,a primary amine at the reducing terminus to facilitate conjugation to aprotein carrier and attachment to microarrays and other surfaces. In thefollowing synthesis, the linker comprises the (CH₂)₅NH₂ group and theoverall synthesis was performed according to scheme 5 or 8 above asindicated.

General Experimental

Commercial grade reagents and solvents were used without furtherpurification except as indicated below. All batch reactions conductedunder an Ar atmosphere. ¹H-NMR and ¹³C-NMR spectra were measured with aVarian 400-MR or Varian 600 spectrometer. The proton signal of residual,non-deuterated solvent (δ 7.26 ppm for CHCl₃; δ 4.79 ppm for H₂O, 2.84ppm for acetone) was used as an internal reference for ¹H spectra. For¹³C spectra, the chemical shifts are reported relative to the respectivesolvent (δ 77.16 ppm for CDCl₃, δ 29.84 ppm for acetone). For ¹³Cspectra in D₂O, MeOH (δ 49.50 ppm) was added as internal standard.Coupling constants are reported in Hertz (Hz). The followingabbreviations are used to indicate the multiplicities: s, singlet; d,doublet; t, triplet; m multiplet. Infrared (IR) spectra were recorded asthin films on a Perkin Elmer Spectrum 100 FTIR spectrophotometer.Optical rotations (OR) were measured with a Schmidt & Haensch UniPol L1000 at 589 nm and a concentration (c) expressed in g/100 mL.High-resolution mass spectra (HRMS) were recorded with an Agilent 6210ESI-TOF mass spectrometer at the Freie Universität Berlin, MassSpectrometry Core Facility. MALDI-TOF spectra were recorded on a BrukerDaltonics Autoflex Speed. Synthetic carbohydrates were measured using a2,4,6-trihydroxyacetophenone (THAP) matrix, proteins and glycoconjugateswere measured using 2,4-dihydroxyacetophenone (DHAP) as matrix.

Analytical thin layer chromatography (TLC) was performed on Kieselgel 60F254 glass plates precoated with a 0.25 mm thickness of silica gel. TheTLC plates were visualized with UV light and by staining with Hanessiansolution (ceric sulfate and ammonium molybdate in aqueous sulfuric acid)or a 1:1 mixture of H₂SO₄ (2N) and resorcine monomethylether (0.2%) inethanol. Column chromatography was performed using Kieselgel 60 (230-400mesh). SEC-HPLC analyses were performed on a TSKgel-G4000SWXL columnconnected to an Agilent 1200 HPLC system equipped with a PDA detector.Elution buffer was constituted by 100 mM sodium phosphate pH 7.2, 100 mMNaCl flow rate was 0.4 mL/min. SDS PAGE gels were run with 10% SDS PAGEgel in reducing conditions at 130 V and 50 mA, molecular weight marker(Invitrogen bench marker) was used.

Synthesis of Pentasaccharide 1 and Intermediates According to Schemes1-5Ethyl-3,4,6-tri-O-benzyl-2-O-(2-naphthalenylmethyl)-1-thio-D-glucopyranoside(7)

To a solution of 6 (284 mg, 0.57 mmol) in anhydrous DMF (1 mL), NaH(20.7 mg, 0.86 mmol) followed by NAP-Br (228 mg, 1.03 mmol) were addedat 0° C. The mixture was warmed to room temperature over 1 h, cooled to0° C. and quenched by the addition of MeOH (0.1 mL). Et₂O was added andthe organic layer washed with 0.01 M HCl solution and with saturatedaqueous NaHCO₃ solution. The phases were separated and the organic layerwas dried over MgSO₄ and concentrated. Column chromatography(hexanes/ethyl acetate) afforded 7 (335 mg, 0.53 mmol, 92%) in a mixtureof α/β-anomers as a white solid. Analytical data is reported for theβ-anomer. [α]_(D) ²⁰=+26.1° (c=5.3, CHCl₃), IR ν_(max) (film) 3061,3030, 2864, 1949, 1808, 1603, 1497, 1453, 1360, 1065 cm⁻¹; ¹H-NMR (400MHz, CDCl₃) δ 7.82-7.69 (4H, m, Ar—H), 7.52-7.09 (18H, m, Ar—H),5.08-5.02 (1H, m, —CH₂—Ar), 4.93-4.77 (4H, m, —CH₂—Ar), 4.60-4.50 (3H,m, —CH₂—Ar), 4.47 (1H, d, J 9.7, 1-H), 3.80-3.54 (4H, m), 3.52-3.41 (2H,m), 2.84-2.66 (2H, m, S—CH₂—), 1.31 (3H, t, J 7.3, CH₃); ¹³C-NMR (100MHz, CDCl₃) δ 138.7, 138.4, 138.2, 135.6, 133.4, 133.2, 128.56, 128.55,128.5, 128.2, 128.1, 127.92, 127.87, 127.84, 127.80, 127.77, 127.7,127.2, 126.5, 126.1, 126.0, 86.8, 85.2 (C-1), 82.0, 79.3, 78.2, 75.9,75.7, 75.2, 73.6, 69.3, 25.2, 15.3; HRMS (ESI): Calcd for C₄₀H₄₂O₅S[M+Na]⁺ 657.2651. found 657.2651.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-3,4,6-tri-O-benzyl-β-D-glucopyranoside(2)

Thioglucoside 7 (335 mg, 0.53 mmol) and HO(CH₂)₅NBnCbz (518 mg, 1.58mmol) were coevaporated with toluene (3×10 ml), dried in vacuo, then thecompounds were dissolved in a solution of anhydrous toluene:dioxane=2:1(4.5 ml). The solution was cooled to −40° C., treated with NIS (131 mg,0.58 mmol) and TfOH (4.7 μl, 53 μmol) and warmed to −20° C. over 1.5 h.The reaction was quenched with pyridine, diluted with DCM and washedwith saturated aqueous Na₂S₂O₃ solution. The organic layer was driedover MgSO₄ and concentrated. Column chromatography on silica gel(hexanes/ethyl acetate) gave a mixture of anomers which was dissolved inDCM (10 ml) and water (1 ml) and treated with DDQ (202 mg, 0.89 mmol) at0° C. for 2 h. The mixture was diluted with DCM and the organic layerwashed with saturated aqueous NaHCO₃ solution, dried over MgSO₄ andconcentrated. Column chromatography on silica gel (hexanes/ethylacetate) afforded 2 (140 mg, 0.184 mmol, 35%) as a colorless oil.[α]_(D) ²⁰=+53.3° (c=5.5), IR ν_(max)·(film) 3458, 3031, 2927, 1952,1876, 1808, 1454, 1421, 1360, 1229, 1129, 1067 cm⁻¹; ¹H-NMR (400 MHz,acetone-d6) δ 7.48-7.10 (25H, m, Ar—H), 5.15 (2H, bs), 4.99 (1H, d, J11.4, —CH₂—Bn), 4.84 (1H, d, J 11.1, —CH₂—Bn). 4.79 (1H, d, J 11.4,—CH₂—Bn), 4.75 (1H, bs, 1-H), 4.62-4.49 (5H, m, —CH₂—Bn), 3.84-3.86 (6H,m), 3.62-3.47 (2H, m), 3.40 (1H, m), 3.31-3.18 (2H, m, linker-CH₂—),1.67-1.50 (4H, m, linker-CH₂—), 1.43-1.29 (2H, m, linker-CH₂—); ¹³C-NMR(100 MHz, acetone-d6) δ 140.5, 139.8, 139.7, 139.5, 129.3, 129.1, 129.0,128.9, 128.60, 128.58, 128.43, 128.41, 128.2, 128.0, 99.9 (C-1), 84.3,78.7, 75.5, 75.4, 74.2, 73.3, 71.5, 70.2, 68.5, 67.4, 24.1; HRMS (ESI):Calcd for C₄₇H₅₃NO₈ [M+Na]⁺ 782.3669. found 782.3633.

(2-Methyl-5-tert-butylphenyl)2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (9)

1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose 8 (30 g, 77 mmol) wasdissolved in anhydrous DCM (34 mL). 2-Methyl-5-tert-butyl thiophenol (17mL, 92 mmol, 1.2 eq) were added under stirring. BF₃.OEt₂ (13.6 mL, 108mmol, 1.4 eq) was added dropwise and the resulting yellow solution wasstirred over night. After completion the solution was diluted with DCMand extracted with saturated aqueous NaHCO₃ and H₂O, and the organiclayer was dried over MgSO₄. The solvent was evaporated in vacuo and theresidue was dried in high vacuum. The resulting yellow solid waspurified by column chromatography on silica gel (cyclohexane/ethylacetate) to afford 9 (33.4 g, 65.4 mmol, 85%). [α]_(D) ²⁰=−8.0° (c=1.0,CHCl₃); IR (CHCl₃): 2961, 1747, 1366, 1211, 1034, 912 cm⁻¹; ¹H-NMR (400MHz, CDCl₃) δ 7.52 (1H, d, J 2.0, Ar—H), 7.25-7.10 (2H, m, Ar—H), 5.19(1H, dd, J₁J₂ 9.4, 1-H), 5.10-4.98 (2H, m, 4-H, 2-H), 4.64 (1H, d, J10.6, 1-H), 4.23 (1H, dd, J₁ 12.2, J₂ 5.0, 6-Ha), 4.10 (1H, dd, J₁12.2,J₂ 1.9, 6-Hb), 3.71-3.63 (1H, m, 5-H), 2.34 (3H, s, CH₃), 2.07-2.03 (6H,m, OAc), 2.00-1.96 (6H, m, OAc), 1.29 (9H, s, tBu); ¹³C-NMR (100 MHz,CDCl₃) δ 170.8, 170.3, 169.5, 169.4 (C═O OAc), 149.8, 137.51, 131.47,130.53, 130.2, 125.8, 87.0 (C-1), 75.9 (C-5), 74.2 (C-3), 70.3 (C-3),68.3 (C-4), 62.4 (C-6), 31.4 (tBu), 20.89, 20.88, 20.74, 20.70 (OAc),20.5 (CH₃); HRMS (ESI): Calcd for C₂₅H₃₄O₉S [M+Na]⁺ 533.1816. found533.1832.

(2-Methyl-5-tert-butylphenyl)-4,6-O-benzylidene-1-thio-β-D-glucopyranoside(10)

Thioglycoside 9 (1.5 g, 2.94 mmol) was dissolved in of methanol (12 mL).Sodium methoxide (58 mg, 1.07 mmol, 0.37 eq) was added and the reactionwas stirred over night. After completion, the solution was neutralizedwith Amberlite IR 120 (H⁺) ion exchange resin, filtered and concentratedin vacuo. The remainder was dried in high vacuum to give(2-Methyl-5-tert-butylphenyl) 1-thio-β-D-glucopyranoside S1 (1.0 g)which was used for the next reaction step without further purification.Tetrol S1 (1.0 g) was dissolved in anhydrous acetonitrile (11.3 mL) atRT under argon atmosphere and benzaldehyde dimethylacetal (880 μL, 5.84mmol, 2 eq) and camphorsulfonic acid (7 mg, 0.029 mmol, 0.01 eq) wereadded. After 2.5 h (TLC: cyclohexane/ethyl acetate, 1:2), the reactionwas quenched with triethylamine, and the solvents were evaporated invacuo to give 1.5 g of colorless oil. The crude product was purified bycolumn chromatography on silica gel (cyclohexane/ethyl acetate) toafford 10 (1.09 g, 2.53 mmol, 87%). [α]_(D) ²⁰=−49.4° (c=1.0, CH₂Cl₂);IR (CH₂Cl₂): 3410, 2963, 2870, 1384, 1264, 1082, 1072, 1029, 1003, 972cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 7.61 (1H, d, J 2.0 Hz, Ar—H), 7.51-7.46(2H, m, Ar—H), 7.39-7.35 (m, 3H, Ar—H), 7.29-7.23 (m, 2H, Ar—H), 7.16(1H, d, J=8.0, Ar—H), 5.54 (1H, s, benzylidene-H), 4.64 (1H, d, J 10.0,1-H), 4.36 (1H, dd, J₁ 10.3, J₂ 4.5, 6-Ha), 3.90-3.73 (2H, m, 3-H,6-Hb), 3.59-3.47 (3H, m, 2-H, 4-H, 5-H), 2.86 (1H, d, J 2.2, OH), 2.69(1H, d, J 2.4, OH), 2.42 (3H, s, CH₃), 1.32 (9H, s, t-Bu); ¹³C-NMR (100MHz, CDCl₃) δ 149.7, 137.1, 137.0, 131.0, 130.3, 130.2, 129.4, 128.5,126.4, 125.5 (C-aromatic), 102.0 (C-benzylidene), 88.8 (C-1), 80.4(C-2), 74.8 (C-3), 73.0 (C-4), 70.5 (C-5), 68.7 (C-6), 31.4 (tBu), 20.6(CH₃); HRMS (ESI): Calcd for C₂₄H₃₀O₅S [M+Na]⁺ 453.1706. found 453.1714.

(2-Methyl-5-tert-butylphenyl)-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside(11)

Compound 10 (658 mg, 1.53 mmol) and imidazole (208 mg, 3.06 mmol, 2 eq)were dissolved in anhydrous DMF (880 μL). TBSCl (346 mg, 2.29 mmol, 1.5eq) was gradually added with stirring. After 4 h, the solvent wasevaporated and the resulting oil was dissolved in DCM. The solution wasextracted with 1 M HCl and saturated aqueous NaHCO₃ solution, theorganic layer was dried over MgSO₄ and the solvent was evaporated invacuo. The colorless solid was dried in high vacuum and the crudeproduct (820 mg) was purified using flash column chromatography(cyclohexane/ethyl acetate) to afford 11 (573 mg, 1.05 mmol, 69%).[α]_(D) ²⁰=−49.1° (c=1.0, CH₂Cl₂); IR (CH₂Cl₂): 3559, 2957, 2928, 2858,1631, 1383, 1259, 1110, 1086, 1067, 1009 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ7.61 (1H, d, J 2.1, Ar—H), 7.51-7.46 (2H, m, Ar—H), 7.39-7.33 (3H, m,Ar—H), 7.26-7.22 (1H, m, Ar—H), 7.15 (1H, d, J 8.0, Ar—H), 5.52 (1H, s,benzylidene-H), 4.65 (1H, d, J 9.8, 1-H), 4.34 (1H, dd, J₁ 10.4, J₂ 4.4,6-Ha), 3.84-3.74 (2H, m, 6-Hb, 3-H), 3.54-3.45 (3H, m, 4-H, 5-H, 2-H),2.42 (3H, s, CH₃), 1.31 (9H, s, tBu), 0.88 (9H, s, tBu), 0.11 (3H, s,CH₃), 0.04 (3H, s, CH₃); ¹³C-NMR (100 MHz, CDCl₃) δ 149.7, 137.3, 137.0,131.4, 130.1, 130.1, 129.1, 128.3, 126.3, 125.3 (C-aromatic), 101.8(C-benzylidene), 89.0 (C-1), 81.2 (C-4), 76.2 (C-3), 74.0 (C-2), 70.8(C-5), 68.8 (C-6), 31.4 (tBu), 26.0 (tBu), 20.6 (CH₃), −4.2 (CH₃), −4.6(CH₃); HRMS (ESI): Calcd for C₃₀H₄₄O₅SSi [M+Na]⁺ 567.2571. found567.2584.

(2-Methyl-5-tert-butylphenyl)4,6-O-benzylidene-2-O-benzyl-1-thio-β-D-glucopyranoside (12)

To a solution of 11 (2.00 g, 3.67 mmol) in anhydrous DMF (20 ml), NaH(0.21 g, 8.81 mmol) and BnBr (1.31 ml, 11.01 mmol) were added at 0° C.The mixture was warmed to room temperature and stirred over night. Thencooled to 0° C., quenched with MeOH and diluted with Et₂O. The organiclayers were washed with H₂O and brine, dried over MgSO₄ andconcentrated. Column chromatography on silica gel (hexanes/ethylacetate) afforded crude (2-methyl-5-tert-butylphenyl)4,6-O-benzylidene-2-O-benzyl-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranosideS2 (2.4 g), which was taken directly to the next step. Crude S2 (2.4 g)was dissolved in THF (30 ml), cooled to 0° C. and treated with asolution of TBAF (1 M in THF, 7.24 ml, 7.24 mmol). The mixture waswarmed to room temperature over night and concentrated. Columnchromatography on silica gel (hexanes/ethyl acetate) afforded 12 (1.77g, 3.40 mmol, 93%). [α]_(D) ²⁰=−11.4° (c=3.7, CHCl₃), IR ν_(max) (film)3463, 3033, 2962, 1810, 1670, 1602, 1488, 1455, 1384, 1264, 1215, 1088cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 7.64-7.61 (1H, m, Ar—H), 7.51-7.20 (11H,m, Ar—H), 7.17-7.12 (1H, m, Ar—H), 5.55 (1H, s, benzylidene-H), 4.99(1H, d, A of AB, J_(AB) 10.9, —CH₂—Bn), 4.84 (1H, d, B of AB, J_(AB)10.9, —CH₂—Bn), 4.75 (1H, d, J 9.8, 1-H), 4.34 (1H, dd, J₁ 10.5, J₂ 5.0,6-Ha), 3.97-3.89 (1H, m, 3-H), 3.81 (1H, dd, J₁ J₂ 10.3, 6-Hb), 3.60(1H, dd, J₁ J₂ 9.4, 4-H), 3.55-3.42 (2H, m, 2-H, 5-H), 2.52 (1H, d, J2.4, 3-OH), 2.42 (3H, s, CH₃), 1.31 (9H, s, tBu); ¹³C-NMR (100 MHz,CDCl₃) δ 149.7, 138.1, 137.1, 136.3, 132.8, 130.1, 129.4, 129.1, 128.7,128.5, 128.4, 128.2, 126.4, 125.0, 102.0, 88.2 (C-1), 81.1 (C-2), 80.5(C-4), 75.7, 75.6 (C-3), 70.1 (C-5), 68.8 (C-6), 34.6, 31.5, 20.5; HRMS(ESI): Calcd for C₃₁H₃₆O₅S [M+Na]⁺ 543.2181. found 543.2181.

(2-Methyl-5-tert-butylphenyl)4,6-O-benzylidene-2-O-benzyl-3-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside(13)

To a solution of 12 (415 mg, 0.80 mmol) and pyridine (129 μl) in DCM (5ml), Fmoc-Cl (309 mg, 1.20 mmol) was added and the mixture was stirredover night, diluted with DCM and the organic layers were washed with a0.01 M HCl solution and saturated aqueous NaHCO₃ solution. The organiclayer was dried over MgSO₄ and concentrated. Column chromatography onsilica gel (hexanes/ethyl acetate) afforded 13 (561 mg, 0.76 mmol, 95%)as a white solid. [α]_(D) ²⁰=−0.3° (c=5.9, CHCl₃), IR ν_(max) (film)3033, 2961, 1955, 1754, 1605, 1451, 1385, 1251, 1077 cm⁻¹; ¹H-NMR (400MHz, CDCl₃) δ 7.79-7.73 (2H, m, Fmoc-H), 7.65-7.13 (19H, m, Ar—H), 5.55(1H, s, benzylidene-H), 5.29-5.22 (1H, m), 4.98 (1H, A of AB, J_(AB)10.7, —CH₂—Bn), 4.82 (1H, d, J 9.8, H-1), 4.72 (1H, B of AB, J 10.7,—CH₂—Bn), 4.49-4.42 (1H, m), 4.40-4.28 (2H, m), 4.24-4.18 (1H, m),3.88-3.67 (3H, m), 3.60-3.52 (1H, m), 2.42 (3H, s, CH₃), 1.31 (9H, s,tBu); ¹³C-NMR (100 MHz, CDCl₃) δ 154.6, 149.8, 143.5, 143.3, 141.4,137.5, 136.9, 136.6, 130.2, 129.6, 129.2, 128.4, 128.3, 128.2, 128.00,127.97, 127.30, 127.27, 126.3, 126.2, 125.2, 120.1, 101.6, 88.7 (C-1),79.5, 79.3, 78.5, 75.7, 70.33, 70.27, 68.8, 46.8, 34.6, 31.4, 20.5; HRMS(ESI): Calcd for C₄₆H₄₆O₇S [M+Na]⁺ 765.2862. found 765.2886.

(2-Methyl-5-tert-butylphenyl)-2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside(14)

To a solution of 13 (100 mg, 0.14 mmol) in anhydrous DCM (3 ml) freshlyactivated molecular sieves (4 Å) were added. The mixture was cooled to−78° C., TES (64 μl, 0.40 mmol) and TfOH (41 μl, 0.46 mmol) were added.After stirring for 3 hours at −78° C. the reaction was quenched by theaddition of pyridine, diluted with DCM and washed with a saturatedaqueous NaHCO₃ solution. The organic phase was then dried over MgSO₄,filtered and concentrated. Column chromatography on silica gel(hexanes/ethyl acetate) afforded 14 (73 mg, 0.10 mmol, 73%). [α]_(D)²⁰=+10.5° (c=4.9, CHCl₃), IR ν_(max) (film) 3486, 3031, 2959, 1951,1750, 1604, 1451, 1387, 1254, 1054 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ7.80-7.74 (2H, m, Fmoc-H), 7.66-7.56 (3H, m, Ar—H), 7.44-7.09 (16H, m,Ar—H), 4.95 (1H, dd, J₁ J₂ 9.2, 3-H), 4.92 (1H, d, J 10.7, —CH₂—Bn),4.69 (1H, d, J 9.8, 1-H), 4.68 (1H, d, J 10.8, —CH₂—Bn), 4.61 (1H, A ofAB, J_(AB) 12.0, —CH₂-Bn), 4.55 (1H, B of AB, J_(AB) 12.0, —CH₂—Bn),4.50-4.43 (1H, m, Fmoc-CH₂), 4.40-4.31 (1H, m, Fmoc-CH₂), 4.26-4.20 (1H,m, Fmoc-CH), 3.84 (1H, ddd, J₁ J₂ 9.5, J₃ 3.6, 4-H), 3.81-3.74 (2H, m,6-H), 3.61 (1H, dd, J₁ J₂ 9.5, 2-H), 3.56-4.49 (1H, m, 5-H), 2.97 (1H,d, J 3.6, 4-OH), 2.40 (1H, s, CH₃), 1.26 (9H, s, tBu); ¹³C-NMR (100 MHz,CDCl₃) δ 155.7, 149.8, 143.5, 143.4, 141.4, 137.7, 137.6, 136.5, 132.8,130.1, 129.5, 128.6, 128.4, 128.2 128.04, 127.98, 127.9, 127.3, 125.3,125.2, 125.0, 120.2, 88.1 (C-1), 83.2 (C-3), 78.5 (C-2), 77.8 (C-5),75.4, 73.9, 71.0 (C-4), 70.4, 70.3 (C-6), 46.9, 34.6, 31.4, 20.5; HRMS(ESI): Calcd for C₄₆H₄₈O₇S [M+Na]⁺ 767.3018. found 767.3038.

(2-Methyl-5-tert-butylphenyl)-2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-4-O-levulinoyl-1-thio-β-D-glucopyranoside(3)

To a solution of 14 (480 mg, 0.64 mmol) in DCM (8 ml) and pyridine (0.3ml) Lev₂O (55 mg, 0.26 mmol) was added and stirred for three days. Themixture was diluted with DCM and washed with a 1 M HCl solution and withsaturated aqueous NaHCO₃ solution. The organic layers were dried overMgSO₄ and concentrated. Column chromatography on silica gel(hexanes/ethyl acetate) afforded 3 (428 mg, 0.51 mmol, 79%). [α]_(D)²⁰=+19.2° (c=1.0, CHCl₃), IR ν_(max) (film) 3065, 2955, 1754, 1719,1604, 1488, 1452, 1363, 1259, 1152, 1070, 1039 cm⁻¹; ¹H-NMR (400 MHz,CDCl₃) δ 7.80-7.74 (2H, m, Ar—H), 7.68-7.58 (3H, m, Ar—H), 7.44-7.17(15H, m, Ar—H), 7.15-7.11 (1H, m, Ar—H), 5.20 (1H, dd, J₁ J₂ 9.7, 4-H),5.15-5.07 (1H, m, 3-H), 4.95 (1H, A of AB, J_(AB) 10.8, —CH₂—Bn), 4.71(1H, d, J 9.8, 1-H), 4.69 (1H, B of AB, J_(AB) 10.4, —CH₂—Bn), 4.56-4.41(3H, m), 4.29-4.20 (2H, m), 3.74-3.55 (4H, m, 2-H, 4-H, 6-H), 2.60-2.52(2H, m, Lev-CH₂), 2.42 (3H, s, Lev-CH₃), 2.41-2.32 (2H, m, Lev-CH₂),2.02 (3H, s, SPhCH₃), 1.26 (9H, s, tBu); ¹³C-NMR (100 MHz, CDCl₃) δ206.0, 171.6, 154.8, 149.9, 143.7, 143.5, 141.4, 141.3, 138.0, 137.6,136.6, 132.7, 130.1, 129.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.7,127.4, 127.3, 125.5, 125.4, 125.0, 120.1, 88.2, 80.5, 78.9, 77.3, 75.6,73.7, 70.6, 69.4, 69.2, 46.7, 37.8, 34.6, 31.4, 29.7, 28.0, 20.5; HRMS(ESI): Calcd for C₅₁H₅₄O₉S [M+Na]⁺ 865.3386 found 865.3412.

(2-Methyl-5-tert-butylphenyl)2-O-benzoyl-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside(15)

Thioglycoside 12 (1.00 g, 1.84 mmol) was dissolved under argon inanhydrous pyridine (4 mL). DMAP (67 mg, 0.55 mmol) was added and thesolution was cooled to 0° C. BzCl (639 μL, 5.51 mmol) was added dropwiseand the solution was heated to 70° C. and stirred for 12 h. Aftercompletion (TLC: cyclohexane/ethyl acetate, 9:1), the reaction wasquenched with methanol. The suspension was diluted with DCM andextracted with 1 M HCl and H₂O. Column chromatography on silica gel(hexanes/ethyl acetate) afforded 15 (1.05 g, 1.62 mmol, 88%). [α]_(D)²⁰=+22.9° (c=1.0, CH₂Cl₂); IR (CH₂Cl₂): 2959, 2929, 2858, 1732, 1384,1266, 1096, 1069 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 8.08 (2H, dd, J 8.3,Ar—H), 7.56 (1H, d, J 1.8, Ar—H), 7.52-7.43 (5H, m, Ar—H), 7.37 (3H, dd,J₁ 5.2, J₂ 2.0, Ar—H), 7.20 (1H, dd, J₁ 8.0, J₂ 2.1, Ar—H), 7.07 (1H, d,J 8.0, Ar—H), 5.58 (1H, s, benzylidene-H), 5.35 (1H, dd, J₁ 10.3, J₂8.6, 2-H), 4.84 (1H, d, J 10.3, 1-H), 4.38 (1H, dd, J₁ 10.5, J₂ 5.0,6-Ha), 4.06 (1H, dd, J₁ J₂ 8.9, 3-H), 3.88 (1H, dd, J₁ 10.3, J₂ 5.0,6-Hb), 3.69 (1H, dd, J₁ J₂ 9.1 Hz, 4-H), 3.60-3.52 (1H, m, 5-H), 2.18(3H, s, CH₃), 1.28 (9H, s, tBu), 0.70 (9H, s, tBu), −0.05 (3H, s, CH₃),−0.14 (3H, s, CH₃); ¹³C-NMR (100 MHz, CDCl₃) δ 133.1, 129.9, 129.8,129.4, 129.1, 128.3, 128.1, 126.2, 125.1 (C—Ar), 101.9 (C-benzylidene),88.1 (C-1), 81.3 (C-4), 74.3 (C-3), 73.6 (C-2), 70.6 (C-5), 68.7 (C-6),31.3 (tBu), 25.5 (tBu), 20.2 (CH₃), −4.2 (CH₃), −5.0 (CH₃); HRMS (ESI):Calcd for C₃₇H₄₈O₆ SSi [M+Na]⁺ 671.2833. found 671.2852.

(2-Methyl-5-tert-butylphenyl)2-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (16)

To a solution of 15 (200 mg, 0.31 mmol) in DMF (1 mL) a solution ofTBAF.3H₂O (683 mg, 1.85 mmol) and glacial acetic acid (124 μL, 2.16mmol) in DMF (1 mL) were added. The mixture was warmed to 35° C. for 9h, diluted with ether and washed with a 0.01 M HCl solution andsaturated aqueous NaHCO₃ solution. The organic layer was dried overMgSO₄ and concentrated. Column chromatography on silica gel(hexanes/ethyl acetate) afforded 16 (150 mg, 0.28 mmol, 91%). [α]_(D)²⁰=−5.5° (c=0.8, CHCl₃); IR (CHCl₃): 3455, 2963, 2870, 1729, 1268, 1100,1071 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 8.11 (2H, d, J 7.4, Ar—H),7.64-7.33 (9H, m, Ar—H), 7.27-7.20 (1H, m, Ar—H), 7.10 (1H, d, J 8.0,Ar—H), 5.59 (1H, s, benzylidene-H), 5.25 (1H, dd, J₁10.1, J₂ 8.7, 2-H),4.88 (1H, d, J 10.1, 1-H), 4.40 (1H, dd, J₁ 10.5, J₂ 5.0, 6-Ha), 4.09(1H, dd, J₁ 9.0, J₂=8.7, 3-H), 3.87 (1H, dd, J₁ 10.4, J₂ 5.0, 6-Hb),3.71 (1H, dd, J₁ 9.0, J₂ 9.7, 4-H), 3.57 (1H, td, J₁ 9.7, J₂ 5.0, 5-H),2.83 (1H, br, 3-OH), 2.23 (3H, s, CH₃), 1.29 (9H, s, tBu); ¹³C-NMR (100MHz, CDCl₃) δ 166.1 (C═O benzoyl), 149.7, 137.32, 136.9, 133.6, 131.9,130.4, 130.2, 129.5, 128.6, 128.5, 126.4, 125.6 (aromatics), 102.1(C-benzylidene), 87.5 (C-1), 80.9 (C-4), 74.0 (C-3), 73.6 (C-2), 70.5(C-5), 68.7 (C-6), 31.4 (tBu), 20.4 (CH₃); HRMS (ESI): Calcd forC₃₁H₃₄O₆S [M+Na]⁺ 557.1968. found 557.1975.

(2-Methyl-5-tert-butylphenyl)2-O-benzoyl-4,6-O-benzylidene-3-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside(17)

To a solution of 16 (277 mg, 0.52 mmol) and pyridine (130 μl) in DCM (4ml), Fmoc-Cl (268 mg, 1.04 mmol) was added and the mixture stirred overnight, diluted with DCM and the organic layers were washed with a 0.01 MHCl solution and saturated aqueous NaHCO₃ solution. The organic layerwas dried over MgSO₄ and concentrated. Column chromatography on silicagel (hexanes/ethyl acetate) afforded 17 (378 mg, 0.50 mmol, 96%).[α]_(D) ²⁰=+50.2° (c=4.5, CHCl₃), IR ν_(max) (film) 3066, 2961, 1752,1732, 1602, 1488, 1450, 1385, 1316, 1268, 1250, 1093 cm⁻¹; ¹H-NMR (400MHz, CDCl₃) δ 8.06-7.99 (2H, m, Ar—H), 7.73-7.67 (2H, m, Ar—H),7.61-7.07 (19H, m, Ar—H), 5.60 (1H, s, benzylidene-H), 5.51-5.36 (2H, m,2-H, 3-H), 4.95 (1H, d, J 9.9, 1-H), 4.46-4.39 (1H, m, 6-H), 4.27-4.16(2H, m, Fmoc-CH₂), 4.06-4.00 (1H, m, Fmoc-CH), 3.98-3.88 (2H, m, 4-H,6-H), 3.72-3.63 (1H, m, 5-H) 2.23 (1H, s, CH₃), 1.29 (9H, s, tBu);¹³C-NMR (100 MHz, CDCl₃) δ 165.3, 154.6, 149.8, 143.4, 143.2, 141.3,141.2, 137.4, 136.8, 133.6, 131.7, 130.5, 130.2, 130.1, 129.3, 129.2,128.5, 128.3, 127.9, 127.27, 127.25, 126.3, 125.8, 125.3, 125.1, 120.00,119.99, 101.8, 88.0 (C-1), 78.3 (4-H), 77.3 (C-3), 71.4 (C-2), 70.8(C-5), 70.5, 68.7 (C-6), 46.6, 34.6, 31.7, 31.4, 20.4, 14.3; HRMS (ESI):Calcd for C₄₆H₄₄O₈S [M+Na]⁺ 779.2655. found 779.2649.

Dibutyl-2-O-benzoyl-4,6-O-benzylidene-3-O-fluorenyl-methoxycarbonyl-D-gluco-pyranosidephosphate(4)

Thioglucoside 17 (690 mg, 0.91 mmol) was coevaporated with toluene threetimes and dried in vacuo, then dissolved in anhydrous DCM (10 ml).Freshly activated molecular sieves (4 Å) and dibutyl hydrogen phosphate(542 μl, 2.73 mmol) were added and the solution cooled to 0° C. NIS (246mg, 1.09 mmol), followed by TfOH (10 μl, 0.11 mmol) was added andstirred at 0° C. for one hour. The reaction was quenched by the additionof pyridine, diluted with DCM and washed with aqueous Na₂S₂O₃ andsaturated aqueous NaHCO₃ solutions. The organic phase was dried overMgSO₄, filtered and concentrated. The crude product was purified bycolumn chromatography on silica gel (hexanes/ethyl acetate) to afford 4(583 mg, 0.74 mmol, 81%) in a mixture of α/β-anomers (α/β=1:4). NMR dataare reported for the β-anomer. [α]_(D) ²⁰=+8.9° (c=3.1, CHCl₃), IRν_(max) (film) 3067, 2961, 1755, 1733, 1602, 1451, 1268, 1096, 1026cm⁻¹; H-NMR (400 MHz, CDCl₃) δ 8.06-7.99 (2H, m, Ar—H), 7.72-7.66 (2H,m, Ar—H), 7.55-7.29 (12H, m, Ar—H), 7.18-7.11 (2H, m, Ar—H), 5.60-5.54(2H, m, benzylidene-H, 1-H), 5.50 (1H, dd, J₁ J₂ 9.4, 2-H), 5.36 (1H,dd, J₁ J₂ 9.4, 3-H), 4.49-4.41 (1H, m, 6-H), 4.30-4.18 (2H, m,Fmoc-CH₂), 4.10-4.01 (3H, m, Fmoc-H, phosphate-CH₂), 4.00-3.94 (1H, m,4-H), 3.90-3.86 (1H, m, 6-H), 3.82-3.67 (3H, m, phosphate-CH₂, 5-H),1.67-1.60 (2H, m, phosphate-CH₂), 1.42-1.25 (4H, m, phosphate-CH₂),1.10-1.01 (2H, m, phosphate-CH₂), 0.92 (3H, t, J 7.4, phosphate-CH-3),0.70 (3H, t, J 7.4, phosphate-CH₃); ¹³C-NMR (100 MHz, CDCl₃) δ 165.1,154.5, 143.4, 143.1, 141.3, 136.6, 133.8, 130.1, 129.4, 128.6, 128.4,127.9, 127.2, 126.3, 125.3, 125.2, 120.0, 101.9, 96.91, 96.86, 78.1,77.5, 77.2, 76.8, 75.8, 72.6, 70.6, 68.4, 68.1, 67.1, 46.6, 32.2, 32.1,32.0, 31.9, 18.7, 18.4, 13.7, 13.5; δ_(P) (160 MHz, CDCl₃) −2.95; HRMS(ESI): Calcd for C₄₃H₄₇O₁₂P [M+Na]⁺ 809.2703. found 809.2690.

4-Methoxyphenyl-2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (19)

Rhamnoside 18 (500 mg, 1.39 mmol) was dissolved in a solution of DCM (1ml) and pyridine (1 ml). DMAP (68 mg, 0.56 mmol) was added and themixture cooled to 0° C., then BzCl (780 mg, 5.56 mmol) was added and thereaction warmed to room temperature over night. The reaction wasquenched with MeOH, diluted with DCM and the organic layer was washedwith a 0.01 M HCl solution and saturated aqueous NaHCO₃ solution. Theorganic layer was dried over MgSO₄ and concentrated. Columnchromatography on silica gel (hexanes/ethyl acetate) afforded 19 (768 g,1.35 mmol, 97%). [α]_(D) ²⁰=+17.6° (c=3.1, CHCl₃), IR ν_(max) (film)3064, 2934, 1725, 1602, 1506, 1452, 1363, 1273, 1213, 1094, 1027 cm⁻¹;¹H-NMR (400 MHz, CDCl₃) δ 8.11-8.05 (2H, m, Ar—H), 7.98-7.93 (2H, m,Ar—H), 7.67-7.61 (1H, m, Ar—H), 7.56-7.49 (3H, m, Ar—H), 7.40-7.35 (2H,m, Ar—H), 7.25-7.16 (5H, m, Ar—H), 7.08-7.03 (2H, m, Ar—H), 6.87-6.82(2H, m, Ar—H), 5.94 (1H, dd, J₁ 9.6, J₂ 3.4, 3-H), 5.79 (1H, dd, J₁ 3.4,J₂1.9, 2-H), 5.54 (1H, d, J 1.8, 1-H), 4.75 (1H, A of AB, J_(AB) 10.9,—CH₂—Bn), 4.68 (1H, B of AB, J_(AB) 10.9, —CH₂—Bn), 4.20-4.11 (1H, m,5-H), 3.88 (1H, dd, J₁ J₂ 9.6, 4-H), 3.78 (3H, s, —CH₃), 1.41 (3H, d, J6.2, 6-H); ¹³C-NMR (100 MHz, CDCl₃) δ 165.58, 165.55, 155.21, 150.20,137.7, 133.6, 133.3, 130.0, 129.9, 129.8, 129.71, 128.69, 128.53,128.48, 128.2, 128.0, 117.9, 114.7, 96.6 (C-1), 79.1 (C-4), 75.3, 72.3(C-3), 71.2 (C-2), 68.5 (C-5), 55.8, 18.3 (C-6); HRMS (ESI): Calcd forC₃₄H₃₂O₈ [M+Na]⁺ 591.1995. found 591.1985.

2,3-Di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside-N-phenyl-trifluoroacetimidate(5)

CAN (2.17 g, 3.96 mmol) was added to a mixture of 19 (750 mg, 1.32 mmol)in MeCN (12 ml) and H₂O (12 ml) and stirred vigorously for 2 h. H₂O andEtOAc were added, the layers separated, the organic layer washed withH₂O and brine, dried over MgSO₄ and concentrated. Column chromatographyon silica gel (hexanes/ethyl acetate) afforded the lactol as an orangesolid (548 mg). A solution of the lactol (548 mg) in DCM (10 ml) wascooled to 0° C., CF₃C(NPh)Cl (438 mg, 2.11 mmol) and Cs₂CO₃ (688 mg,2.11 mmol) were added and the resulting solution was stirred overnightat room temperature, diluted with DCM, filtered through a plug of celiteand concentrated. Column chromatography on silica gel (hexanes/ethylacetate) afforded 5 (619 mg, 0.98 mmol, 74%). [α]_(D) ²⁰=+41.2° (c=4.8,CHCl₃), IR ν_(max) (film) 3065, 2981, 1727, 1600, 1490, 1452, 1270,1208, 1164, 1091 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 8.07-8.02 (2H, m,Ar—H), 7.96-7.89 (2H, m, Ar—H), 7.66-7.60 (1H, m, Ar—H), 7.57-7.46 (3H,m, Ar—H), 7.40-7.19 (9H, m, Ar—H), 7.40-7.19 (9H, m, Ar—H), 7.14-7.07(1H, m, Ar—H), 6.91-6.82 (2H, m, Ar—H), 6.35 (1H, bs, 1-H), 5.84 (1H, s,2-H), 5.77 (1H, dd, J₁9.4, J₂ 3.3, 3-H), 5.35 (1H, dd, J₁ 3.7, J₂ 1.9,1-H), 4.76 (1H, A of AB, J_(AB) 10.9, —CH₂—Bn), 4.68 (1H, B of AB,J_(AB) 10.9, —CH₂-Bn), 4.21-4.08 (1H, m, 5-H), 3.87 (1H, dd, J₁ J₂ 9.5,4-H), 1.48 (3H, d, J 6.1, 6-H); ¹³C-NMR (100 MHz, CDCl₃) δ 165.5, 165.3,143.4, 137.4, 133.7, 133.4, 130.0, 129.8, 129.7, 129.4, 128.9, 128.7,128.58, 128.57, 128.3, 128.2, 124.6, 119.6, 94.1 (C-1), 78.5 (C-4),75.5, 72.0 (C-3), 70.7 (C-3), 69.6 (C-2), 18.4 (C-6); HRMS (ESI): Calcdfor C₃₅H₃₀F₃NO₇ [M+Na]⁺ 656.1872, found 656.1852.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-4-O-levulinoyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside(20)

Glucoside donor 3 (326 mg, 0.34 mmol) and glucoside acceptor 2 (262 mg,0.35 mmol) were coevaporated with toluene three times and dried invacuo. The mixture was dissolved in anhydrous Et₂O (3 ml), NIS (93 mg,0.41 mmol) was added and cooled to −35° C. TfOH (3.7 μl, 41 μmol) wasadded and the mixture was stirred and warmed up to −10° C. in one hour.The reaction was quenched by the addition of pyridine, diluted with DCMand washed with aqueous Na₂S₂O₃ and saturated aqueous NaHCO₃ solutions.The phases were separated and the aqueous phase was extracted with DCM.The combined organic phases were dried over MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography onsilica gel (hexanes/ethyl acetate) to afford 20 (343 mg, 0.24 mmol,70%). [α]_(D) ²⁰=+64.4° (c=5.9), IR ν_(max) (film) 3032, 2932, 1755,1700, 1605, 1497, 1452, 1362, 1259 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ8.00-6.90 (43H, m, Ar—H), 5.41 (1H, dd, J₁ J₂ 9.7), 5.26 (1H, dd, J₁ J₂9.8), 5.18-5.10 (2H, m), 5.06 (1H, bs, anomeric-H), 5.03-4.96 (2H, m,anomeric-H), 4.88 (1H, app d, J 11.0), 4.82 (1H, app d, J 10.8),4.68-4.58 (3H, m), 4.52-4.41 (5H, m), 4.39-4.30 (2H, m), 4.26 (1H, appt, J 7.5), 4.14-4.08 (1H, m), 4.07-4.01 (1H, m), 3.82 (1H, dd, J₁ 9.9,J₂ 3.4), 3.80-3.56 (6H, m), 3.34-3.31 (2H, m), 3.28-3.06 (4H, m),2.54-2.42 (2H, m), 2.32-2.17 (2H, m), 2.00 (1H, s, Lev-CH₃), 1.65-1.50(4H, m, linker-CH₂—), 1.30-1.23 (4H, m, linker-CH₂—); ¹³C-NMR (100 MHz,CDCl₃) δ 206.0, 171.5, 154.9, 143.7, 143.6, 141.40, 141.35, 138.0,137.8, 128.6, 128.54, 128.53, 128.39, 128.37, 128.2, 128.1, 128.0,127.94, 127.87, 127.74, 127.66, 127.5, 127.3, 126.3, 125.5, 120.1,120.0, 95.6 (C-anomeric), 94.0 (C-anomeric), 80.7, 78.2, 77.4, 77.0,76.8, 76.2, 75.9, 75.4, 73.7, 73.5, 72.4, 70.5, 70.3, 68.8, 68.6, 68.4,67.3, 46.8, 37.8, 31.4, 29.8, 27.9, 23.7; HRMS (ESI): Calcd forC₈₇H₉₁NO₁₇ [M+Na]⁺ 1444.6179. found 1444.6128.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside(21)

To a solution of 20 (224 mg, 0.16 mmol) in DCM (4.5 ml) hydrazinehydrate (31 μl, 0.63 mmol) dissolved in AcOH (0.4 ml) and pyridine (0.6ml) was added and the solution stirred for 1 h. The reaction was thenquenched by the addition of acetone and concentrated. Columnchromatography on silica gel (hexanes/ethyl acetate) afforded 21 (196mg, 0.15 mmol, 94%). [α]_(D) ²⁰=+57.7° (c=1.7), IR ν_(max) (film) 3423,3031, 2926, 1753, 1697, 1605, 1586, 1497, 1452, 1422, 1362, 1255, 1068cm⁻¹; ¹H-NMR (400 MHz, acetone-d6) δ 7.92-7.84 (2H, m, Ar—H), 7.78-7.64(2H, m, Ar—H), 7.56-7.14 (35H, m, Ar—H), 5.44-5.37 (2H, m), 5.20-5.10(3H, m), 5.07 (1H, d, J 10.7), 4.89-4.77 (3H, m), 4.66-4.47 (8H, m),4.46-4.39 (2H, m), 4.27 (1H, app t, J 6.9), 4-20-4.14 (1H, m), 3.99 (1H,app t, J 9.3), 3.89-3.80 (2H, m), 3.78-3.59 (7H, m), 3.59-3.52 (1H, m),3.49-3.42 (1H, m), 3.25-3.15 (2H, m), 2.82-2.79 (1H, m), 1.60-1.44 (4H,m, linker-CH₂—), 1.33-1.25 (2H, m, linker-CH₂—); ¹³C-NMR (100 MHz,acetone-d6) δ 155.9, 144.7, 144.6, 142.2, 142.1, 139.9, 139.8, 139.74,139.68, 139.5, 139.4, 129.34, 129.26, 129.1, 129.02, 129.00, 128.9,128.7, 128.62, 128.55, 128.5, 128.4, 128.20, 128.16, 128.14, 128.05,128.0, 127.9, 126.1, 126.0, 120.88, 120.86, 96.3, 94.2, 81.8, 80.0,79.2, 78.0, 76.5, 75.5, 73.8, 73.5, 72.1, 71.9, 71.5, 70.2, 70.08,70.06, 69.5, 68.6, 67.4, 47.6, 27.5, 24.2; HRMS (ESI): Calcd forC₈₂H₈₅NO₁₅[M+Na]⁺ 1346.5817. found 1346.5784.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside(23)

Phosphate 4 (74 mg, 94 μmol) and 21 (48 mg, 36 μmol) were coevaporatedwith toluene three times, dried in vacuo and then dissolved in anhydrousDCM (1.0 ml). Freshly activated molecular sieves (4 Å) were added andthe mixture cooled to −30° C. TMSOTf (18 μl, 98 μmol) was added and thenwarmed to −7° C. over 1.5 h. The reaction was quenched with pyridine andconcentrated in vacuo. Column chromatography on silica gel(toluene/acetone) afforded crude 22. 20% NEt₃ in DCM (1 ml) was added tocrude 22 and stirred for 4 h, the mixture was concentrated in vacuocolumn chromatography on silica gel (toluene/acetone) afforded 23 (20mg, 14 μmol, 38%). [α]_(D) ²⁰=+8.1° (c=1.6), IR ν_(max) (film) 3462,3032, 2924, 1732, 1699, 1603, 1497, 1453, 1364, 1268, 1093 cm⁻¹; ¹H-NMR(400 MHz, CDCl₃) δ 8.05-7.92 (2H, m, Ar—H), 7.63-7.06 (43H, m, Ar—H),5.56 (1H, s, benzylidene-H), 5.24 (1H, app t, J 8.5), 5.20-5.11 (2H, m),5.09-4.98 (2H, m, anomeric-H), 4.88 (1H, app d, J 10.7), 4.79-4.66 (4H,m, anomeric-H), 4.62-4.54 (1H, m), 4.49-4.36 (5H, m), 4.19-4.05 (2H, m),4.03-3.91 (2H, m), 3.89-3.44 (14H, m), 3.39-3.04 (4H, m), 1.57-1.36 (4H,m, linker-CH₂—), 1.32-1.14 (2H, m, linker-CH₂—); ¹³C-NMR (100 MHz,CDCl₃) δ 165.6, 138.51, 138.46, 138.4, 138.1, 136.8, 133.7, 130.1,129.6, 129.4, 128.7, 128.6, 128.54, 128.52, 128.47, 128.45, 128.4,127.98, 127.9, 127.84, 127.78, 127.7, 127.4, 126.4, 102.1, 101.7(C-anomeric), 95.8 (C-anomeric), 94.5 (C-anomeric), 81.1, 80.8, 80.6,78.2, 77.9, 77.4, 76.1, 75.2, 74.7, 73.6, 73.4, 72.8, 72.1, 71.6, 70.4,69.5, 68.7, 68.4, 68.2, 67.8, 67.3, 66.4, 29.8, 29.4, 23.6; HRMS (ESI):Calcd for C₈₇H₉₃NO₁₉ [M+Na]⁺ 1478.6239. found 1478.6136.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(13)-2-O-benzoyl-4,6-O-benzylidene-3-β-D-glucopyranosyl-(1→4)-[2,3-Di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)]-2,6-di-O-benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside(24)

Compounds 5 (26 mg, 41 μmol) and 23 (10 mg, 6.9 μmol) were coevaporatedwith toluene three times, dried in vacuo and dissolved in anhydrous DCM(1.0 ml). Freshly activated molecular sieves (4 Å) were added and themixture cooled to −30° C. TMSOTf (10 μl of a solution of 7.4 μl TMSOTfin 93 μl DCM, 4.1 μmol) was added and the reaction was stirred at −30°C. for 1.5 h. The reaction was quenched with pyridine and concentratedin vacuo. Column chromatography on silica gel (toluene/acetone) afforded24 (14 mg, 5.5 μmol, 81%). [α]_(D) ²⁰=+5.2° (c=0.7), IR ν_(max) (film)3032, 2933, 1728, 1602, 1585, 1496, 1452, 1363, 1263, 1094, 1069 cm⁻¹;¹H-NMR (400 MHz, CDCl₃) δ 8.20-6.90 (75H, m, Ar—H), 5.79-5.67 (3H, m),5.46 (1H, s, benylidene-H), 5.33-5.29 (1H, m), 5.28-5.21 (1H, m),5.17-5.08 (3H, m, anomeric-H), 5.02 (1H, bs, anomeric-H), 4.92-4.78 (4H,m, anomeric-H), 4.74-4.60 (4H, m), 4.59-4.49 (4H, m, anomeric-H),4.48-4.44 (1H, m), 4.43-4.31 (4H, m), 4.29-4.13 (4H, m, anomeric-H),4.03-3.88 (3H, m), 3.83-3.45 (13H, m), 3.40-3.02 (7H, m), 1.65 (1H, d, J6.2, Rha-CH₃), 1.53-1.32 (4H, m, linker-CH₂—), 1.24-1.10 (2H, m,linker-CH₂—), 0.90 (1H, d, J 6.1, Rha-CH₃); ¹³C-NMR (100 MHz, CDCl₃) δ165.6, 165.48, 165.5, 164.5, 164.2, 138.3, 137.8, 137.6, 133.1, 130.1,130.0, 129.94, 129.85, 129.7, 129.4, 129.1, 129.0, 128.9, 128.83,128.76, 128.7, 128.6, 128.51, 128.47, 128.45, 128.42, 128.36, 128.32,128.29, 128.23, 128.17, 128.04, 128.00, 127.94, 127.88, 127.8, 127.7,126.5, 126.4, 100.6 (C-anomeric), 100.5 (C-anomeric), 97.9 (C-anomeric),97.5, 95.8 (C-anomeric), 93.5 (C-anomeric), 80.2, 79.2, 78.1, 77.5,77.4, 77.2, 76.8, 76.2, 76.1, 76.0, 74.2, 74.0, 73.6, 72.9, 72.1, 71.6,71.2, 70.9, 68.7, 67.4, 67.2, 50.6, 47.2, 46.2, 29.9, 23.6, 18.4, 17.5;HRMS (ESI): Calcd for C₁₄₁H₁₄₁NO₃₁ [M+Na]⁺ 2366.9385. found 2366.9440.

5-Amino-pentanylα-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→3)]-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside(1)

Fully protected pentasaccharide 24 (10 mg, 4.3 μmol) was dissolved in asolution of NaOMe (0.5 M) in THF/MeOH (1:1, 1 ml) and heated to 50° C.for 12 h. The mixture was neutralized with Amberlite IR 120 (H⁺) ionexchange resin, filtered and concentrated. Size exclusion chromatographyon Sephadex LH-20 (CHCl₃/MeOH=1:1) afforded the de-benzoylatedpentasaccharide (5.6 mg), which was dissolved in a mixture of MeOH (0.9ml), H₂O (0.1 ml) and AcOH (25 μl). The solution was purged with Argon,10% Pd/C (10 mg) was added and the solution purged with H₂ for 30 min,then stirred under an H₂ atmosphere for 12 h, filtered and concentrated.Size exclusion chromatography on Sephadex LH-20 (MeOH) afforded 1 (2.3mg, 2.6 μmol, 61%). NMR data are reported in Table 1, comparison withthe data from native PS-I is reported in Table 2. HRMS (MALDI-TOF):Calcd for C₃₅H₆₃NO₂₄ [M+Na]⁺ 904.3632. found 904.3606.

TABLE 1 ¹H NMR δ (600 MHz, D₂O) and ¹³C NMR δ (150 MHz, D₂O) ofpentasaccharide 1.^(a) α-Glc α-Glc β-Glc α-Rha α-Rha (A) (B) (C) (D)(D′) Linker H-1 5.18 5.09 4.53 5.24 5.14 C-1 96.1 96.8 102.4 101.8 102.0H-2 3.70 3.73 3.38 4.06 4.06 C-2 72.7 73.4 75.3 71.4 71.2 H-3 3.70 4.033.61 3.88 3.81 C-3 76.1 77.0 83.2 71.1 71.2 H-4 3.48 3.86 3.46 3.47 3.47C-4 70.5 73.8 69.1 73.0 73.0 H-5 3.82 4.05 3.45 4.43 4.03 C-5 72.5 72.377.2 69.5 69.8 H-6 3.88/3.78 3.92 3.80/3.96 1.27 1.27 a/b C-6 61.6 60.362.2 17.5 17.5 H-1′ 3.79/3.59 a/b C-1′ 68.7 H-2′ 1.70 C-2′ 29.0 H-3′1.49 C-3′ 23.5 H-4′ 1.70 C-4′ 27.7 H-5′ 3.01 C-5′ 40.4 ^(a1)H and ¹³CNMR resonances were assigned based on HSQC, HMBC, COSY and TOCSYexperiments.

TABLE 2 Comparison of ¹H and ¹³C NMR δ between 1 and the native PS-Irepeating unit.^(a) α-Glc α-Glc β-Glc α-Rha α-Rha (A) (B) (C) (D) (D′)H-1  5.18  5.09  4.53 5.24 5.14  5.75  5.13  4.53 5.23 5.17 C-1 96.196.8 102.4  101.8   102.0   93.5 98.0 102.4  101.9   101.4   H-2  3.70 3.73  3.38 4.06 4.06  3.68  3.70  3.38 4.07 4.09 C-2 72.7 73.4 75.371.4  71.2  77.3 73.6 75.2 71.1  71.2  H-3  3.70  4.03  3.61 3.88 3.81 3.89  4.01  3.62 3.85 3.97 C-3 76.1 77.0 83.2 71.1  71.2  72.1 77.583.0 71.0  70.9  H-4  3.48  3.86  3.46 3.47 3.47  3.53  3.86  3.46 3.464.07 C-4 70.5 73.8 69.1 73.0  73.0  70.1 73.6 69.1 73.0  78.9  H-5  3.82 4.05  3.45 4.43 4.03  3.91  4.06  3.45 4.44 4.12 C-5 72.5 72.3 77.269.5  69.8  73.8 72.4 77.1 69.4  68.6  H-6 3.88/3.78  3.92 3.80/3.961.27 1.27 a/b n.d. n.d. 3.80/3.95 1.27 1.33 C-6 61.6 60.3 62.2 17.5 17.5  n.d. n.d. 62.2 17.5  17.8  ^(a)data of native PS-I reported initalic taken from: J. Ganeshapillai et al., Carbohydr. Res., 2008, 343,703.

Synthesis of Pentasaccharide 1 and Intermediates According to Schemes6-8 (2-Methyl-5-tert-butylphenyl)4,6-O-benzylidene-2-O-benzyl-3-O-(4-bromo)benzyl-1-thio-β-D-glucopyranoside(25)

To a solution of 12 (200 mg, 0.38 mmol) in anhydrous DMF (2 ml), NaH (22mg, 0.92 mmol) was added followed by para-bromobenzyl (PBB) bromide (288mg, 1.15 mmol) at 0° C. The mixture was warmed to room temperature over2 h, cooled to 0° C. and quenched by the addition of MeOH. Et₂O wasadded and the organic layer washed with 0.1 M HCl solution and withsaturated aqueous NaHCO₃ solution. The phases were separated and theorganic layer was dried over MgSO₄ and concentrated. Columnchromatography (cyclohexane/ethyl acetate) afforded 25 (276 mg) alongwith aromatic impurities and was taken to the next step without furtherpurification.

(2-Methyl-5-tert-butylphenyl)2,6-di-O-benzyl-3-O-(4-bromo)benzyl-1-thio-β-D-glucopyranoside (26)

To a solution of 25 (140 mg, 0.20 mmol) in anhydrous DCM (4 ml) freshlyactivated molecular sieves (4 Å) were added. The mixture was cooled to−78° C., TES (97 μl, 0.61 mmol) and TfOH (61 μl, 0.69 mmol) were added.After stirring for 3 hours at −78° C., the reaction was quenched by theaddition of saturated aqueous NaHCO₃ solution, diluted with DCM andwashed with a saturated aqueous NaHCO₃ solution. The organic phase wasthen dried over MgSO₄, filtered and concentrated. Column chromatographyon silica gel (cyclohexane/ethyl acetate) afforded 26 (81 mg, 0.12 mmol,58%). ¹H-NMR (400 MHz, CDCl₃) δ 7.65-7.60 (m, 1H, ArH), 7.54-7.10 (m,16H, ArH), 4.98 (d, 1H, J=10.3 Hz, benzyl), 3.65-3.44 (m, 6H, benzyl,1-H), 3.79-3.70 (m, 3H, 6-H, 4-H), 3.56-3.43 (m, 3H, 2-H, 3-H, 5-H),2.76 (d, 1H, J=2.2 Hz, 4-OH), 2.40 (s, 3H, CH₃), 1.26 (s, 9H, tBu);¹³C-NMR (100 MHz, CDCl₃) δ 149.7, 138.1, 137.72, 137.67, 136.1, 133.3,131.7, 130.0, 129.6, 128.9, 128.6, 128.5, 128.3, 128.0, 128.0, 124.7,121.8, 88.2 (C-1), 86.2 (C-2), 80.7 (C-3), 77.5 (C-5), 75.7, 74.7, 73.9,72.7 (C-4), 70.7 (C-6), 31.4, 20.5; HRMS (ESI): Calcd for C₃₈H₄₃BrO5SNa⁺[M+Na]⁺ 713.1907. found 713.1951.

(2-Methyl-5-tert-butylphenyl)2,6-di-O-benzyl-3-O-(4-bromo)benzyl-4-O-levulinoyl-1-thio-β-D-glucopyranoside(27)

To a solution of 26 (1.55 g, 2.24 mmol) in DCM (20 ml) at 0° C., DMAP(274 mg, 2.24 mmol), LevOH (1.30 ml, 11.20 mmol) and DCC (2.31 g, 11.20mmol) were added. The solution was warmed to room temperature andstirred for 16 h. The reaction was diluted with DCM and the organiclayers were washed with a 0.1 M HCl solution and saturated aqueousNaHCO₃ solution. The organic layer was dried over MgSO₄ andconcentrated. Column chromatography on silica gel (hexanes/ethylacetate) afforded 27 (1.54 g, 1.95 mmol, 87%). [α]_(D) ²⁰=+6.4° (c=3.4,CHCl₃), IR ν_(max) (film) 2963, 1744, 1718, 1488, 1361, 1261, 1068,1038, 1012 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 7.70-7.05 (m, 17H, Ar—H),5.11-5.04 (m, 1H, 4-H), 4.97 (app. d, 1H, J=10.4 Hz, benzyl-H),4.77-4.60 (m, 4H, benzyl-H, 1-H), 4.48 (s, 1H, PBB-H), 3.70-3.54 (m, 5H,2-H, 3-H, 5-H, 6-H), 2.64-2.55 (m, 2H, Lev-CH₂), 2.40 (s, 3H, S—CH₃),2.35-2.29 (m, 2H, Lev-CH₂), 2.12 (s, 3H, Lev-CH₃), 1.25 (s, 9H, tBu);¹³C-NMR (100 MHz, CDCl₃) δ 206.2 (Lev-carbonyl), 171.7, 149.8, 138.1,138.0, 137.5, 136.2, 133.2, 131.6, 130.0, 129.6, 128.9, 128.5, 128.4,128.3, 128.1, 128.0, 127.69, 124.71, 121.6, 88.3 (C-1), 84.1, 81.1,77.4, 75.8, 74.5, 73.7, 71.3, 69.7, 37.8, 34.6, 31.4, 29.9, 28.0, 20.5;HRMS (MALDI-TOF): Calcd for C₄₃H₄₉BrO₇SNa⁺ [M+Na]⁺ 811.2275. found811.2026.

(2-Methyl-5-tert-butylphenyl)2-O-benzoyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside(28)

To a solution of 15 (800 mg, 1.23 mmol) in anhydrous DCM (12 ml) freshlyBH₃.THF (1 M in THF, 7.4 ml, 7.4 mmol) and TMSOTf (0.11 ml, 0.62 mmol)were added drop wise at 0° C. The reaction was warmed to roomtemperature over 2 hours, cooled to 0° C. again and quenched by the dropwise addition of saturated aqueous NaHCO₃ solution. The Emulsion wasdiluted with DCM and washed with a saturated aqueous NaHCO3 solution.The organic phase was then dried over MgSO4, filtered and concentrated.Crude 28 was taken to the next step.

(2-Methyl-5-tert-butylphenyl)2-O-benzoyl-4,6-di-O-benzyl-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside(29)

To a solution of crude 28 (approx. 1.23 mmol) in THF/DMF (9:1, 10 ml) at0° C., BnBr (0.18 ml, 1.50 mmol) and NaH (36 mg, 1.50 mmol) were added.The solution was warmed to room temperature over 2 h, then cooled to 0°C. again and further BnBr (0.18 ml, 1.50 mmol) was added. The reactionwas warmed to room temperature over 30 min, cooled to 0° C. and quenchedby the addition of water. After dilution with Et₂O the phases wereseparated and the aqueous layer extracted with Et₂O. The organic phasewas then dried over MgSO4, filtered and concentrated. Columnchromatography on silica gel (hexanes/ethyl acetate) afforded 29 (797mg, 1.08 mmol, 88%). ¹H-NMR (400 MHz, CDCl₃) δ 8.14-7.00 (m, 18H, Ar—H),5.31 (dd, 1H, J₁=10.1 Hz, J₂=8.9 Hz, 2-H), 4.83 (app. d, 1H, J=11.3 Hz,benzyl-Ha), 4.72 (d, 1H, J=10.2 Hz, 1-H), 4.63 (app. d, 1H, J=11.0 Hz,benzyl-H_(b)), 4.58 (app. d, 2H, J=3.1 Hz, benzyl-H), 3.95 (app. t, 1H,J=8.7 Hz, 3-H), 3.78-3.51 (m, 4H, 4-H, 5-H, 6-H), 2.15 (s, 3H, S—CH₃),1.25 (s, 9H, S-tBu), 0.79 (s, 9H, TBS-tBu), 0.00 (s, 3H, TBS-CH₃), −0.16(s, 3H, TBS-CH₃); ¹³C-NMR (100 MHz, CDCl₃) δ 165.6, 149.7, 138.2, 136.5,133.2, 130.5, 130.1, 129.8, 129.2, 128.5, 128.4, 128.0, 127.72, 127.68,127.6, 124.7, 88.0 (C-1), 79.5 (C-5), 78.9 (C-4), 77.0 (C-3), 75.1, 73.6(C-2), 73.5, 69.0 (C-6), 31.4, 25.8, 20.3, −3.9, −4.1; HRMS (ESI): Calcdfor C₄₄H₅₆O₆SSiNa⁺ [M+Na]⁺ 763.3459. found: 763.3500

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-Tri-O-benzyl-α-D-glucopyranoside(30)

Thioglucoside 27 (323 mg, 0.41 mmol) and glucoside 2 (222 mg, 0.29 mmol)were coevaporated with toluene three times and dried in vacuo. Themixture was dissolved in Ether (4 ml), freshly activated and acid washedmolecular sieves (4 Å) and NIS (105 mg, 0.47 mmol) were added and cooledto −40° C. TfOH (4.2 μl, 0.05 mmol) was added and the mixture wasstirred and warmed up to −10° C. in one hour. The reaction was quenchedby the addition of pyridine, diluted with DCM and washed with aqueousNa₂S₂O₃ and saturated aqueous NaHCO₃ solutions. The phases wereseparated and the aqueous phase was extracted with DCM. The combinedorganic phases were dried over MgSO₄, filtered and concentrated. Thecrude product was purified by column chromatography on silica gel(toluene/acetone) to afford 30 (276 mg, 0.20 mmol, 69%). [α]_(D)²⁰=+54.1° (c=4.8, CHCl₃), IR ν_(max) (film) 3031, 2923, 2864, 1744,1698, 1497, 1454, 1420, 1360, 1209 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ7.60-7.02 (m, 39H, Ar—H), 5.24-5.10 (m, 3-H), 5.09-4.93 (m, 3H, 2×anomeric-H), 4.89-4.37 (m, 13H), 4.13-4.00 (m, 2H), 3.99-3.56 (m, 8H),3.50-3.08 (m, 5H), 2.63-2.47 (m, 2H), 2.25-2.18 (m, 2H), 2.13 (s, 3H,Lev-CH₃), 1.71-1.38 (m, 4H, linker-H), 1.36-1.14 (m, 2H, linker-H);¹³C-NMR (100 MHz, CDCl₃) δ 206.3 (Lev-carbonyl), 171.4, 138.7, 138.3,138.1, 138.1, 137.8, 131.4, 129.6, 128.7, 128.5, 128.4, 128.3, 128.1,128.03, 127.98, 127.93, 127.88, 127.8, 127.60, 127.57, 127.4, 121.4,95.5 (C-anomeric), 93.5 (C-anomeric), 80.9, 79.3, 78.8, 78.1, 75.7,75.3, 74.1, 73.7, 73.5, 72.3, 70.5, 70.2, 68.6, 68.3, 68.1, 67.3, 37.8,30.0, 29.5, 27.9, 23.7; HRMS (MALDI-TOF): Calcd for C₇₉H₈₆BrNO₁₅Na⁺[M+Na]⁺ 1390.5073. found 1390.5105.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-Tri-O-benzyl-α-D-glucopyranoside(31)

To a solution of 30 (300 mg, 0.22 mmol) in DCM (5.0 ml) hydrazinehydrate (32 μl, 0.66 mmol) dissolved in AcOH (0.4 ml) and pyridine (0.6ml) was added and the solution stirred for 1 h. The reaction was thenquenched by the addition of acetone and concentrated. Columnchromatography on silica gel (hexanes/ethyl acetate) afforded 31 (117mg, 0.09 mmol, 96%). [α]_(D) ²⁰=+56.5° (c=2.7, CHCl₃), IR ν_(max) (film)3453, 2963, 1695, 1454, 1420, 1360, 1259, 1013 cm⁻¹; H-NMR (600 MHz,CDCl₃) δ 7.90-7.00 (39H, m, Ar—H), 5.25-5.13 (m, 2H), 5.10 (bs, 1H,anomeric-H), 5.05 (bs, 1H, anomeric-H), 4.98-4.43 (m, 14H), 4.10-3.53(m, 13H), 3.45-3.10 (m, 3H), 1.65-1.40 (m, 4H, linker-H), 1.34-1.15 (m,2H, linker-H); ¹³C-NMR (150 MHz, CDCl₃) δ 138.7, 138.2, 138.1, 131.6,129.7, 128.6, 128.49, 128.45, 128.1, 128.0, 127.97, 127.91, 127.85,127.74, 127.71, 127.3, 121.6, 95.6 (C-anomeric), 93.9 (C-anomeric),81.4, 81.0, 78.9, 78.1, 77.4, 77.2, 77.0, 75.8, 75.2, 74.4, 73.6, 73.6,72.1, 71.1, 70.5, 69.3, 68.6, 68.3, 67.3, 50.3, 47.2, 46.2, 43.3, 29.5,27.7, 23.6; HRMS (MALDI-TOF): Calcd for C₇₄H₈₀BrNO₁₃Na⁺ [M+Na]⁺1292.4705. found 1292.4701.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2-O-benzoyl-4,6-di-O-benzyl-3-O-tert-butyldimethylsilyl-α-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside(32)

Thioglucoside 29 (233 mg, 0.31 mmol) and disaccharide 31 (266 mg, 0.21mmol) were coevaporated with toluene three times and dried in vacuo. Themixture was dissolved in DCM (7 ml), freshly activated and acid washedmolecular sieves (4 Å) and NIS (80 mg, 0.36 mmol) were added and cooledto −30° C. TfOH (3.2 μl, 0.04 mmol) was added and the mixture wasstirred and warmed up to −17° C. in one hour. The reaction was quenchedby the addition of pyridine, diluted with DCM and washed with aqueousNa₂S₂O₃ and saturated aqueous NaHCO₃ solutions. The phases wereseparated and the aqueous phase was extracted with DCM. The combinedorganic phases were dried over MgSO₄, filtered and concentrated. Thecrude product was purified by column chromatography on silica gel(toluene/acetone) to afford 32 (354 mg, 0.19 mmol, 92%). [α]_(D)²⁰=+52.5° (c=2.6, CHCl₃), IR ν_(max) (film) 3031, 2928, 2859, 1733,1699, 1603, 1497, 1454, 1421, 1362, 1314, 1265, 1070 cm⁻¹; ¹H-NMR (400MHz, CDCl₃) δ 7.91-7.05 (m, 54H, Ar—H), 5.21-5.11 (m, 3H), 5.04 (bs, 1H,anomeric-H), 5.01-4.95 (m, 2H, anomeric-H), 4.81 (app. d, 1H, J=11.3Hz), 4.74-4.35 (m, 17H, anomeric-H), 4.23 (app. d, 1H, J=12.3 Hz), 3.98(app. t, 1H, J=9.4 Hz), 3.93-3.87 (m, 1H), 3.82 (app. t, 1H, J=9.3 Hz),3.74-3.66 (m, 4H), 3.64-3.45 (m, 10H), 3.42-3.36 (m, 1H), 3.34-3.06 (m,4H), 1.56-1.35 (m, 4H), 1.25-1.09 (m, 2H), 0.79 (s, 9H, tBu), 0.02 (s,3H), −0.19 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ 164.8, 138.7, 138.7,138.4, 138.4, 138.4, 138.1, 133.1, 131.1, 130.1, 130.0, 129.6, 128.7,128.6, 128.49, 128.48, 128.43, 128.41, 128.40, 128.37, 128.35, 128.2,128.0, 127.93, 127.86, 127.8, 127.7, 127.64, 127.58, 127.5, 127.4,120.8, 100.3 (C-anomeric), 96.1 (C-anomeric), 59.0 (C-anomeric) 80.5,80.0, 79.1, 78.6, 77.7, 76.1, 75.5, 75.4, 75.3, 75.2, 74.7, 74.4, 73.8,73.6, 73.5, 72.3, 70.6, 70.3, 69.1, 68.7, 67.6, 67.2, 50.6, 47.2, 46.3,29.4, 28.1, 25.8, 23.6, 17.9, −3.86, −3.89; HRMS (MALDI-TOF): Calcd forCo₁₀₇H₁₂₀BrNO₁₉SiNa⁺ [M+Na]⁺ 1852.7299 found 1852.7375.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2-O-benzoyl-4,6-di-O-benzyl-β-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-α-D-glucopyranosyl-(12)-3,4,6-tri-O-benzyl-α-D-glucopyranoside(33)

A solution of 32 (100 mg, 0.06 mmol), (3,4-dimethoxyphenyl)boronic acid(20 mg, 0.11 mmol), TBABr (1.8 mg, 5.5 μmol), K₃PO₄ (35 mg, 0.16 mmol)in EtOH (4 ml) was subjected to three freeze-pump-saw cycles. To thissolution Pd(OAc)₂ (1.2 mg, 5.5 μmol) was added and stirred for 2 hours.The mixture was diluted with EtOAc and washed with saturated aqueousNaHCO₃ solution. The aqueous phase was back extracted with EtOAc. Thecombined organic phases were dried over MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography onsilica gel (toluene/acetone) to afford the Suzuki coupling product (95mg, 0.05 mmol, 92%) which was dissolved in DCM/H₂O/saturated aqueousNaHCO₃ (100:9:1, 11 ml). To this emulsion DDQ (34 mg, 0.15 mmol) wasadded, stirred vigorously for 16 hours, diluted with DCM and washed withsaturated aqueous NaHCO₃ solutions. The combined organic phases weredried over MgSO₄, filtered and concentrated. The crude product wasdissolved in DMF (2.5 ml), and treated with a solution of TBAF.3H₂O (137mg, 0.43 mmol) and AcOH (29 μl, 0.51 mmol) in DMF (2.5 ml) at 50° C. forthree days. After dilution with Et₂O the phases were separated and theorganic phase washed with a 0.1 M HCl solution, saturated aqueous NaHCO₃solution and brine. The organic phase was then dried over MgSO4,filtered and concentrated. The crude product was purified by columnchromatography on silica gel (toluene/acetone) to afford 33 (52 mg, 0.03mmol, 68%). [c]D²⁰=+38.9° (c=1.5, CHCl₃), IR ν_(max) (film) 3462, 3031,2924, 2867, 1729, 1699, 1497, 1454, 1422, 1362, 1315, 1268, 1095, 1069cm⁻¹; H-NMR (400 MHz, CDCl₃) δ 8.07-7.00 (m, 50H), 5.25-5.05 (m, 3H),5.03-4.94 (m, 2H, 2× anomeric-H), 4.90 (app. d, J=10.6, 1H), 4.82-4.35(m, 16H), 4.27 (app. d, J=12.1, 1H), 4.16 (app. dd, J=9.2, 8.8, 1H),4.06 (app. d, J=12.2, 1H), 3.99 (app. t, J=9.3, 1H), 3.93-3.42 (m, 15H),3.28 (s, 4H), 1.73-1.36 (m, 4H, linker-H), 1.34-1.08 (m, 2H, linker-H);¹³C-NMR (100 MHz, CDCl₃) δ 166.2, 139.0, 138.5, 138.4, 137.9, 137.7,133.6, 130.1, 129.4, 128.72, 128.65, 128.62, 128.59, 128.57, 128.52,128.46, 128.45, 128.40, 128.36, 128.30, 128.24, 128.18, 127.97, 127.95,127.93, 127.88, 127.8, 127.61, 127.57, 127.37, 101.2 (C-anomeric), 95.9(C-anomeric), 94.8 (C-anomeric), 81.6, 78.4, 78.2, 78.0, 77.5, 77.4,77.2, 76.8, 76.6, 76.3, 75.0, 74.7, 73.9, 73.6, 73.2, 72.7, 72.2, 70.4,69.3, 69.1, 68.7, 67.3, 50.4, 47.3, 29.5, 28.1, 23.6; HRMS (MALDI-TOF):Calcd for C₉₄H₁₀₁NO₁₉Na⁺ [M+Na]⁺ 1570.6860. found 1570.6362.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzyl-β-D-glucopyranosyl-(1→4)-[2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)]-2,6-di-O-benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside(34)

Rhamnosyl-imidate 5 (72 mg, 140 μmol) and trisaccharide 33 (42 mg, 27μmol) were coevaporated with toluene three times, dried in vacuo anddissolved in anhydrous DCM (3.0 ml). Freshly activated molecular sieves(4 Å) were added and the mixture cooled to −40° C. TMSOTf (25 μl of asolution of 100 μl TMSOTf in 900 μl DCM, 14 μmol) was added and thereaction was warmed to −20° C. over 1.5 h. The reaction was quenchedwith TEA and concentrated. Size exclusion chromatography on SephadexLH-20 (CHCl₃/MeOH 1:1) afforded 34 (58 mg, 24 μmol, 88%). [α]_(D)²⁰=+49.7° (c=2.2, CHCl₃), IR ν_(max) (film) 3031, 2927, 2863, 1729,1700, 1602, 1497, 1453, 1273, 1264, 1095, 1069 cm⁻¹; ¹H-NMR (600 MHz,CDCl₃) δ 8.08-6.98 (m, 80, Ar—H), 5.89 (app. dd, J=9.4, 3.5, 1H), 5.85(app. dd, J=3.5, 1.7, 1H), 5.66 (app. dd, J=9.4, 3.5, 1H), 5.49-5.42 (m,1H), 5.39 (app. dd, J=3.5, 1.8, 1H), 5.32-5.25 (m, 1H), 5.16-5.12 (m,2H), 5.085.05 (m, 1H), 5.04-4.99 (m, 1H), 4.97 (d, J=1.6, 1H), 4.954.25(m, 20H), 4.24-3.43 (m, 20H), 3.39-3.00 (m, 5H), 1.67 (d, J=6.2, 3H),1.60-1.32 (m, 4H), 1.32-1.06 (m, 2H), 0.94 (d, J=6.1, 3H); ¹³C-NMR (150MHz, CDCl₃) δ 165.43, 165.21, 164.49, 164.12, 139.19, 138.54, 138.37,138.16, 137.83, 137.74, 133.13, 133.01, 132.95, 132.69, 132.68, 130.38,130.14, 130.11, 129.99, 129.94, 129.89, 129.78, 129.75, 129.74, 129.45,128.95, 128.70, 128.67, 128.65, 128.62, 128.55, 128.47, 128.45, 128.41,128.39, 128.36, 128.26, 128.23, 128.19, 128.15, 128.02, 127.95, 127.93,127.91, 127.75, 127.66, 127.36, 127.22, 99.53, 97.97, 97.73, 95.81,93.74, 80.83, 80.40, 80.26, 79.27, 78.40, 78.25, 77.52, 76.58, 76.15,75.91, 75.74, 75.16, 74.68, 74.20, 74.01, 73.68, 73.58, 73.27, 72.87,72.19, 71.93, 71.20, 71.16, 70.59, 70.33, 68.69, 68.09, 67.99, 67.33,67.22, 50.61, 47.18, 46.26, 29.44, 23.56, 18.58, 17.75; HRMS(MALDI-TOF): Calcd for C₁₄₈H₁₄₉NO₃₁Na⁺ [M+Na]⁺ 2459.0006. found2459.0636.

5-Amino-pentanylα-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→3)]-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside(1)

To a solution of fully protected pentasaccharide 34 (23 mg, 9.4 μmol) inTHF (1.5 ml) NaOMe (0.5 M, in MeOH, 1 ml) was added and stirred for 12h. The mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, filtered and concentrated. Column chromatography on silica gel(DCM/acetone/MeOH) afforded the de-benzoylated pentasaccharide (16 mg),which was dissolved in a mixture of THF (1 ml) MeOH (1 ml), H₂O (0.7 ml)and AcOH (0.1 ml). The solution was purged with Ar, 10% Pd/C (30 mg) wasadded and the solution purged with H₂ for 30 min, then stirred under anH₂ atmosphere for 12 h, filtered and concentrated. Size exclusionchromatography on Sephadex LH-20 (MeOH) afforded 1 (5.0 mg, 5.7 μmol,60%). NMR data is consistent with previously reported.³

EXAMPLE 2 Preparation of PS-I Substructures 5-Amino-pentanylD-glucopyranosyl-(1→2)-α-D-glucopyranoside (35)

A solution of protected disaccharide 33 (40 mg, 31 μmol) in a mixture ofMeOH (5.0 ml), THF (2.5 ml) H₂O (2.0 ml) and AcOH (0.5 ml) was purgedwith Ar. After that 10% Pd/C (70 mg) was added and the solution purgedwith H₂ for 30 min, then stirred under an H₂ atmosphere for 12 h,filtered and concentrated. The crude product was purified by reversedphase solid phase extraction (RP SPE) (Waters Sep-Pak®, C18) to afford35 (13.3 mg, 31 μmol, 99%). ¹H-NMR (600 MHz, D₂O) δ 5.23 (d, J=3.4, 1H,anomeric), 5.16 (d, J=3.6, 1H, anomeric), 4.02-3.80 (m, 8H), 3.75 (app.dd, J=9.9, 3.5, 2H) 3.68-3.61 (m, 2H), 3.53 (app. td, J=9.6, 4.7, 2H),3.09 (app. t, J=7.5, 2H), 1.81-1.71 (m, 4H, linker), 1.59-1.49 (m, 2H,linker); ¹³C-NMR (150 MHz, D₂O) δ 98.6 (anomeric), 97.9 (anomeric),77.7, 75.4, 74.5, 74.4, 74.2, 74.0, 72.3, 72.1, 70.4, 63.3, 63.1, 42.1,30.6, 29.2, 25.1; HRMS (MALDI-TOF): Calcd for C₁₇H₃₃NO₁₁H⁺ [M+H]⁺428.2126. found 428.2147.

5-Amino-pentanylβ-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside(36)

To a solution of protected trisaccharide 33 (60 mg, 31 μmol) in THF (2ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. Themixture was neutralized with Amberlite IR 120 (H+) ion exchange resin,filtered and concentrated. The crude product was dissolved in a mixtureof THF (5.0 ml) MeOH (2.5 ml), H₂O (2.0 ml) and AcOH (0.5 ml). Thesolution was purged with Ar, then 10% Pd/C (30 mg) was added and thesolution purged with H₂ for 30 min, then stirred under an H₂ atmospherefor 12 h, filtered and concentrated. Purification by RP SPE (WatersSep-Pak®, C18) afforded 36 (13.3 mg, 31 μmol, 66%). ¹H-NMR (600 MHz,D₂O) δ 5.22 (d, J=3.3, 1H, anomeric α-Glc), 5.15 (d, J=3.6, 1H, anomericα-Glc), 4.60 (d, J=7.9, 1H, anomeric β-Glc), 4.14-4.08 (m, 1H),4.03-3.91 (m, 5H), 3.90-3.79 (m, 4H), 3.78-3.72 (m, 3H), 3.71-3.62 (m,2H), 3.62-3.47 (m, 4H), 3.40 (t, J=8.7, 1H), 3.09 (t, J=7.5, 2H),1.83-1.72 (m, 4H, linker), 1.59-1.49 (m, 2H, linker). ¹³C-NMR (150 MHz,D₂O) δ 100.7 (anomeric 3-Glc), 94.0 (anomeric α-Glc), 93.4 (anomericα-Glc), 76.8, 74.2, 73.7, 73.5, 71.3, 69.8, 69.6, 69.5, 69.2, 68.7,67.7, 67.6, 65.9, 58.8, 57.9, 37.5, 26.1, 24.6, 20.6; HRMS (MALDI-TOF):Calcd for C₂₃H₄₃NO₁₆Na⁺ [M+Na]⁺ 612.2474. found 612.2424.

(2-Methyl-5-tert-butylphenyl)2-O-benzoyl-4,6-di-O-benzyl-1-thio-β-D-glucopyranoside (40)

A solution of TBAF.3H₂O (1.10 g, 3.48 mmol) and acetic acid (266 μl,4.64 mmol) in DMF (4 ml) was added to a solution of 29 (430 mg, 0.58mmol) in DMF (4 ml). The mixture was stirred for 3 days at 35° C. Afterdilution with Et₂O the phases were separated and the organic phasewashed with a 0.1 M HCl solution, saturated aqueous NaHCO₃ solution andbrine. The organic phase was then dried over MgSO4, filtered andconcentrated. The product 40 was taken directly to the next step.

(2-Methyl-5-tert-butylphenyl)2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)2-O-benzoyl-4,6-di-O-benzyl-1-thio-β-D-glucopyranoside(41)

Rhamnosyl-imidate 5 (373 mg, 0.59 mmol) and glucoside 40 (approx. 0.58mmol) were coevaporated with toluene three times, dried in vacuo anddissolved in anhydrous DCM (3.0 ml). Freshly activated molecular sieves(4 Å) were added and the mixture cooled to −40° C. TMSOTf (10 μl, 53μmol) was added and the reaction was warmed to −20° C. over 1.5 h. Thereaction was quenched with TEA and concentrated. Column chromatographyon silica gel (hexanes/ethyl acetate) afforded 41 (490 mg, 0.46 mmol,79%). [α]_(D) ²⁰=+70.7° (c=1.9, CHCl₃), IR ν_(max) (film) 2963, 1728,1602, 1451, 1259, 1090, 1067, 1025 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ8.02-7.03 (m, 33H), 5.72 (dd, J=9.4, 3.5, 1H), 5.53-5.42 (m, 2H), 5.22(d, J=1.9, 1H), 4.88 (d, J=10.6, 1H), 4.77-4.47 (m, 6H), 4.24-4.13 (m,2H), 3.92-3.80 (m, 3H), 3.68-3.59 (m, 2H), 2.18 (s, 3H), 1.25 (s, 9H),1.08 (d, J=6.2, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 149.8, 138.1, 138.0,133.1, 130.3, 130.0, 129.9, 129.8, 129.7, 129.7, 128.64, 128.57, 128.51,128.46, 128.42, 128.39, 128.37, 128.32, 128.28, 128.24, 128.20, 128.1,128.00, 127.97, 127.9, 127.83, 127.80, 127.75, 125.7, 124.4, 97.6, 86.6,79.3, 77.5, 77.2, 76.8, 75.7, 75.6, 75.0, 74.4, 73.8, 72.0, 71.3, 68.3,67.9, 31.5, 19.5, 18.0; HRMS (MALDI-TOF): Calcd for C₆₅H₆₆O₁₂SNa⁺[M+Na]⁺ 1093.4167. found 1093.4159.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)2-O-benzoyl-4,6-di-O-benzyl-1-thio-β-D-glucopyranoside(42)

Disaccharide 41 (50 mg, 47 μmol) and 5-aminopentanol (31 mg, 93 μmol)were coevaporated with toluene three times and dried in vacuo. Themixture was dissolved in DCM (3 ml) and NIS (13 mg, 56 μmol) was addedand cooled to −20° C. TfOH (0.5 μl, 6 μmol) was added and the mixturewas stirred and warmed up to 0° C. in two hours. The reaction wasquenched by the addition of aqueous Na₂S₂O₃ and saturated aqueousNaHCO₃. The phases were separated and the aqueous phase was extractedwith DCM. The combined organic phases were dried over MgSO₄, filteredand concentrated. The crude product was purified by columnchromatography on silica gel (hexanes/ethyl acetates) to afford 42 (52mg, 43 μmol, 91%). [α]_(D) ²⁰=+50.3° (c=2.6, CHCl₃), IR ν_(max) (film)3032, 2936, 1730, 1698, 1452, 1265, 1069 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ8.23-6.80 (m, 40H, aromatic), 5.73 (dd, J=9.4, 3.5, 1H), 5.46 (dd,J=3.4, 1.9, 1H), 5.35 (dd, J=9.2, 7.9, 1H), 5.24 (d, J=1.9, 1H, anomericRha), 5.14 (bs, 2H), 4.89 (app. d, J=10.6, 1H), 4.72-4.59 (m, 4H),4.56-4.35 (m, 4H, anomeric Glc), 4.22-4.12 (m, 2H), 3.91-3.76 (m, 4H),3.68-3.58 (m, 2H), 3.42-3.33 (m, 1H), 3.05-2.88 (m, 2H), 1.50-1.29 (m,4H, linker), 1.24-0.98 (m, 5H, linker, Rha CH₃). ¹³C-NMR (100 MHz,CDCl₃) δ 165.7, 164.8, 138.2, 138.0, 137.6, 133.1, 132.8, 30.0, 129.92,129.88, 129.8, 129.7, 128.6, 128.5, 128.42, 128.38, 128.36, 128.31,128.30, 128.2, 128.0, 127.94, 127.93, 127.8, 127.7, 101.1 (anomericGlc), 97.6 (anomeric Rha), 79.3, 77.8, 76.9, 75.64, 75.59, 74.9, 74.6,73.8, 71.9, 71.3, 68.9, 68.3, 67.2, 29.2, 23.2, 18.0 (Rha CH₃); HRMS(MALDI-TOF): Calcd for C₇₄H₇₅NO₁₅Na⁺ [M+Na]⁺ 1240.5029. found 1240.4792.

5-Amino-pentanyl α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranoside (38)

To a solution of protected disaccharide 42 (50 mg, 41 μmol) in THF (2ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. Themixture was neutralized with Amberlite IR 120 (H+) ion exchange resin,filtered and concentrated. The crude product was dissolved in a mixtureof THF (5.0 ml) MeOH (2.5 ml), H₂O (2.0 ml) and AcOH (0.5 ml). Thesolution was purged with Ar, then 10% Pd/C (100 mg) was added and thesolution purged with H₂ for 30 min, then stirred under an H₂ atmospherefor 12 h, filtered and concentrated. Purification by RP SPE (WatersSep-Pak®, C18) afforded 38 (15.7 mg, 27 μmol, 78%). ¹H-NMR (600 MHz,D₂O) δ 5.20 (s, 1H, anomeric Rha), 4.53 (d, J=8.1, 1H, anomeric Glc),4.15-4.04 (m, 2H), 4.02-3.96 (m, 2H), 3.85 (app. dd, J=9.7, 3.3, 1H),3.81-3.73 (m, 2H), 3.66 (app. t, J=8.7, 1H), 3.56-3.49 (m, 3H), 3.44 (t,J=8.7, 1H), 3.08 (app. t, J=7.5, 2H), 1.75 (tt, J=14.6, 7.2, 4H,linker), 1.57-1.49 (m, 2H, linker), 1.32 (d, J=6.3, 3H, Rha CH₃);¹³C-NMR (150 MHz, D₂O) δ 100.0 (anomeric Glc), 99.1 (anomeric Rha),80.3, 73.9, 71.8, 70.0, 68.4, 68.2, 68.1, 66.9, 66.2, 58.8, 37.4, 26.2,24.4, 20.1, 14.5 (Rha CH₃); HRMS (MALDI-TOF): Calcd for C₁₇H₃₃NO₁₀Na⁺[M+Na]⁺ 434.1997. found 434.1975.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,6-di-O-benzyl-3-O-(4-bromo)benzyl-4-O-levulinoyl-1-thio-β-D-glucopyranoside(43)

Thioglucoside 27 (300 mg, 0.38 mmol) and 5-aminopentanol (200 mg, 0.61mmol) were coevaporated with toluene three times and dried in vacuo. Themixture was dissolved in Ether (4 ml) and Dioxane (4 ml), NIS (103 mg,0.46 mmol) was added and cooled to −10° C. TfOH (4 μl, 46 μmol) wasadded and the mixture was stirred and warmed up to 0° C. in three hours.The reaction was quenched by the addition of aqueous Na₂S₂O₃ andsaturated aqueous NaHCO₃. The phases were separated and the aqueousphase was extracted with DCM. The combined organic phases were driedover MgSO₄, filtered and concentrated. The crude product was purified bycolumn chromatography on silica gel (hexanes/ethyl acetates) to afford43 (140 mg, 0.15 mmol, 39%). [α]_(D) ²⁰=+22.0° (c=3.4, CHCl₃), IRν_(max) (film) 2920, 1743, 1697, 1454, 1420, 1360, 1208, 1153, 1069,1038 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 7.696.92 (m, 24H, ar), 5.22-5.15(m, 2H), 5.09-5.03 (m, 1H), 4.81 (app. d, J=11.9, 1H), 4.76-4.68 (m, 2H,anomeric), 4.63-4.56 (m, 2H), 4.54-4.46 (m, 4H), 3.89 (app. t, J=9.4,1H), 3.84-3.78 (m, 1H), 3.62-3.45 (m, 4H), 3.38-3.18 (m, 3H), 2.66-2.53(m, 2H), 2.43-2.29 (m, 2H), 2.13 (s, 3H, Lev CH₃), 1.66-1.48 (m, 4H,linker), 1.38-1.27 (m, 2H, linker); ¹³C-NMR (100 MHz, CDCl₃) δ 206.3(Lev carbonyl), 171.6, 138.2, 138.1, 138.0, 131.4, 129.6, 129.4, 128.7,128.5, 128.3, 128.1, 128.03, 127.99, 127.9, 127.6, 127.4, 121.3, 96.9(anomeric), 79.8, 79.6, 74.3, 73.7, 73.2, 70.9, 69.0, 68.9, 68.3, 67.3,37.8, 29.9 (Lev CH₃), 29.2, 28.0, 23.6; HRMS (MALDI-TOF): Calcd forC₅₂H₅₈BrNO₁₀Na⁺ [M+Na]⁺ 958.3134. found 958.3112.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,6-di-O-benzyl-3-O-(4-bromo)benzyl-1-thio-β-D-glucopyranoside (44)

To a solution of 43 (140 mg, 0.15 mmol) in DCM (5.0 ml) hydrazinehydrate (26 μl, 0.54 mmol) dissolved in AcOH (0.4 ml) and pyridine (0.6ml) was added and the solution stirred for 1 h. The reaction was thenquenched by the addition of acetone and concentrated. Columnchromatography on silica gel (hexanes/ethyl acetate) afforded 44 (102mg, 0.12 mmol, 81%). [α]_(D) ²⁰=+24.3° (c=4.2, CHCl₃), IR ν_(max) (film)3454, 3031, 2920, 1696, 1454, 1422, 1229, 1055 cm⁻¹; ¹H-NMR (400 MHz,CDCl₃) δ 7.48-7.04 (m, 24H, Ar), 5.16-5.09 (m, 2H), 4.86 (app. d,J=11.7, 1H), 4.70-4.43 (m, 8H), 3.75-3.55 (m, 6H), 3.45 (app. dd, J=9.5,3.6, 1H), 3.32-3.14 (m, 3H), 1.59-1.44 (m, 4H, linker), 1.33-1.23 (m,2H, linker); ¹³C-NMR (100 MHz, CDCl₃) δ 138.3, 138.1, 138.0, 131.6,129.5, 128.6, 128.52, 128.47, 128.02, 127.98, 127.9, 127.8, 127.7,127.4, 121.6, 96.9 (anomeric), 81.7, 79.8, 74.6, 73.7, 72.9, 71.4, 70.1,69.8, 68.1, 67.3, 50.4, 47.3, 29.2, 27.7, 23.7; HRMS (MALDI-TOF): Calcdfor C₄₇H₅₂BrNO₈Na⁺ [M+Na]⁺ 860.2769. found 860.2508.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzyl-β-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranoside(45)

Disaccharide 41 (144 mg, 0.13 mmol) and glucoside 44 (102 mg, 0.12 mmol)were coevaporated with toluene three times and dried in vacuo. Themixture was dissolved in DCM (4 ml) and NIS (36 mg, 0.16 mmol) was addedand cooled to −20° C. TfOH (1.4 μl, 16 μmol) was added and the mixturewas stirred and warmed up to 0° C. in two hours. The reaction wasquenched by the addition of aqueous Na₂S₂O₃ and saturated aqueousNaHCO₃. The phases were separated and the aqueous phase was extractedwith DCM. The combined organic phases were dried over MgSO₄, filteredand concentrated. The crude product was purified by columnchromatography on silica gel (hexanes/ethyl acetates) to afford 45 (200mg, 0.12 mmol, 95%). [α]_(D) ²⁰=+36.9° (c=5.2, CHCl₃), IR ν_(max) (film)3031, 2866, 1730, 1698, 1602, 1452, 1262, 1092 cm⁻¹; ¹H-NMR (400 MHz,CDCl₃) δ 8.31-6.72 (m, 54H, Ar), 5.73 (app. dd, J=9.4, 3.4, 1H), 5.44(app. dd, J=3.4, 1.9, 1H), 5.37 (app. dd, J=9.3, 8.1, 1H), 5.21 (bs,2H), 5.17-5.09 (m, 2H), 4.88 (app. d, J=10.9, 1H), 4.76-4.39 (m, 13H),4.31 (app. d, J=12.2, 1H), 4.19 (app. dd, J=9.5, 6.1, 1H), 4.03-3.63 (m,9H), 3.49-3.42 (m, 3H), 3.37-3.15 (m, 4H), 1.59-1.40 (m, 4H), 1.28-1.11(m, 5H); ¹³C-NMR (100 MHz, CDCl₃) δ 165.2, 164.6, 164.5, 138.9, 138.5,138.2, 138.0, 137.89, 137.87, 137.6, 133.1, 133.0, 132.9, 131.1, 129.9,129.8, 129.7, 129.63, 129.59, 129.4, 129.2, 128.7, 128.6, 128.5, 128.43,128.35, 128.3, 128.24, 128.19, 128.14, 128.08, 128.0, 127.90, 127.89,127.74, 127.67, 127.61, 127.55, 127.3, 120.7, 100.3 (anomeric), 97.7(anomeric), 96.9 (anomeric), 80.3, 79.1, 78.0, 77.4, 76.7, 75.6, 75.2,74.9, 74.8, 74.5, 73.6, 73.5, 73.1, 71.9, 71.1, 69.7, 68.8, 68.3, 68.0,67.7, 67.2, 29.0, 23.3, 17.9 (Rha CH₃); HRMS (MALDI-TOF): Calcd forC₁₀₁H₁₀₂BrNO₂₀Na⁺ [M+Na]⁺ 1750.6071. found 1759.5921.

5-Amino-pentanylα-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-α-D-glucopyranoside(37)

To a solution of protected trisaccharide 45 (61 mg, 35 μmol) in THF (2ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. Themixture was neutralized with Amberlite IR 120 (H⁺) ion exchange resin,filtered and concentrated. The crude product was dissolved in a mixtureof THF (5.0 ml) MeOH (2.5 ml), H₂O (2.0 ml) and AcOH (0.5 ml). Thesolution was purged with Ar, then 10% Pd/C (100 mg) was added and thesolution purged with H₂ for 30 min, then stirred under an H₂ atmospherefor 12 h, filtered and concentrated. Purification by RP SPE (WatersSep-Pak®, C18) afforded 37 (12.5 mg, 30 μmol, 75%). 1H-NMR (600 MHz,D₂O) δ 5.21 (s, 1H, anomeric Rha), 4.99 (d, J=2.9, 1H, anomeric α-Glc),4.61 (d, J=8.0, 1H, anomeric β-Glc), 4.15-4.05 (m, 2H), 4.02-3.97 (m,2H), 3.93-3.79 (m, 6H), 3.73-3.66 (m, 3H), 3.64-3.49 (m, 5H), 3.09 (t,J=7.1, 2H), 1.81-1.71 (m, 4H, linker), 1.59-1.50 (m, 2H, linker), 1.33(d, J=6.0, 3H, Rha CH₃); ¹³C-NMR (150 MHz, D₂O) δ 102.9 (anomeric Rha),101.7 (anomeric β-Glc), 98.4 (anomeric α-Glc), 82.7, 79.7, 76.5, 74.5,72.6, 72.4, 71.6, 71.1, 71.0, 70.8, 69.4, 68.6, 68.5, 61.2, 60.6, 40.0,28.6, 27.1, 23.0, 17.1. (Rha CH₃); HRMS (MALDI-TOF): Calcd forC₂₃H₄₃BrNO₁₅Na⁺ [M+Na]⁺ 596.2525. found 596.2540.

N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (46)

Rhamnoside-imidate (127 mg, 0.20 mmol) and 5-aminopentanol (160 mg, 0.49mmol) were coevaporated with toluene three times, dried in vacuo anddissolved in anhydrous DCM (3 ml). Freshly activated molecular sieves (4Å) were added and the mixture cooled to −30° C. TMSOTf (3.6 μl, 20 μmol)was added and the reaction was warmed to −20° C. over 1 h. The reactionwas quenched with TEA and concentrated. Column chromatography on silicagel (hexanes/ethyl acetate) afforded 46 (145 mg, 0.19 mmol, 94%).[α]_(D) ²⁰=+54.1° (c=2.6, CHCl₃), IR ν_(max) (film) 2963, 1727, 1260,1018 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 8.28-7.00 (m, 25H, Ar), 5.73 (app.dd, J=9.6, 3.4, 1H), 5.59 (bs, 1H), 5.19 (app d, J=11.3, 2H), 4.87 (bs,1H, anomeric), 4.68 (app. dd, J=28.1, 10.9, 2H), 4.53 (bs, 2H), 3.96(bs, 1H), 3.79 (app. t, J=9.5, 1H), 3.75-3.61 (m, 1H), 3.48-3.21 (m,3H), 1.65-1.51 (m, 4H), 1.45-1.27 (m, 5H); ¹³C-NMR (100 MHz, CDCl₃) δ165.6, 165.5, 138.1, 137.8, 133.4, 133.2, 130.0, 129.9, 129.7, 128.7,128.6, 128.47, 128.46, 128.2, 127.99, 127.95, 127.4, 97.5 (anomeric),79.3, 75.3, 72.6, 71.5, 68.0, 67.8, 67.3, 29.3, 23.6, 18.3; HRMS(MALDI-TOF): Calcd for C₄₇H₄₉NO₉Na⁺ [M+Na]⁺ 794.3300. found 794.3264.

5-Amino-pentanyl α-L-rhamnopyranoside (39)

To a solution of protected rhamnoside 46 (145 mg, 0.19 mmol) in THF (4ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. Themixture was neutralized with Amberlite IR 120 (H⁺) ion exchange resin,filtered and concentrated. The crude product was dissolved in a mixtureof THF (10 ml) MeOH (5 ml), H₂O (4 ml) and AcOH (1 ml). The solution waspurged with Ar, then 10% Pd/C (300 mg) was added and the solution purgedwith H₂ for 30 min, then stirred under an H₂ atmosphere for 12 h,filtered and concentrated. Purification by RP SPE (Waters Sep-Pak®, C18)afforded 39 (44 mg, 0.18 mmol, 94%). ¹H-NMR (600 MHz, D₂O) δ 4.85 (s,1H, anomeric Rha), 4.01-3.96 (m, 1H), 3.81-3.70 (m, 3H), 3.62-3.57 (m,1H), 3.50 (app. t, J=9.6, 1H), 3.11-3.03 (m, 2H), 1.78-1.67 (m, 4H,linker), 1.56-1.46 (m, 2H), 1.34 (d, J=6.3, 3H, Rha CH₃)¹³C-NMR (150MHz, D₂O) δ 98.3 (anomeric), 70.6, 70.0, 68.8, 67.1, 66.1, 38.0, 26.6,25.1, 21.0, 15.2 (Rha CH₃); HRMS (MALDI-TOF): Calcd for C₁₁H₂₃NO₅Na⁺[M+Na]⁺ 272.1468. found 272.1433.

EXAMPLE 3 Preparation and Characterization of an Pentasaccharide-ProteinConjugate

Polysaccharide vaccines provoke exclusively a T-cell independent immuneresponse and do not induce an immunoglobulin class switch. The syntheticrepeating unit 1 of the Clostridium difficile glycopolymer PS-I wasconjugated to the protein carrier Crm₁₉₇. The detoxified diphtheriatoxoid Crm₁₉₇ was chosen as a carrier since it is an approvedconstituent of licensed vaccines (Barocchi et al. (2007), Vaccine 25,2963-73).

Conjugations

A) To a solution of Di(N-succinimidyl) adipate (5.8 mg, 17 μmol) in DMSO(250 μl) and NEt₃ (20 μl) pentasaccharide 1 (500 μg, 0.57 μmol)dissolved in DMSO (250 μl) was added dropwise. The solution was stirredfor 2 h, diluted with phosphate buffer (1.0 ml, 100 μM, pH 7.5) andextracted with CHCl₃. CRM₁₉₇ (rDNA) (250 μl, 250 μg, Pfenex Inc (USA))was added to the aqueous layer and stirred for 5 h. Conjugate 1a wasdesalted and concentrated. An average load of 3.6 pentasaccharide unitsper protein was determined by MALDI-TOF MS, SEC-HPLC and SDS PAGEconfirmed modification of the protein (FIG. 2). SEC-HPLC t_(R)=22.49min, MS (MALDI-TOF) found 61853 Da.

B) First, the primary amine group of the linker moiety of PS-Ipentasaccharide 1 was reacted with one of the ester groups of the spacermolecule di(N-succinimidyl) adipate in water-free DMSO (12.7 mg in 120μl) in the presence of 10 μl triethylamine at room temperature over 2hours, with the spacer used in 10-fold molar excess to avoid dimerformation.

After addition of 400 μL 0.1 M Na-phosphate buffer, pH 7.4, unreactedspacer molecules were removed by chloroform extraction. The remainingester group of the spacer moiety was then reacted with the ε-aminogroups of lysine residues on the CRM₁₉₇ protein (Pfenex) in 0.1 MNa-phosphate buffer, pH 7.4, at room temperature over 12 hours (FIG. 3).For one reaction, 3 mg of PS-I pentasaccharide and 1 mg of CRM₁₉₇(solubilized in 1 mL 0.1 M Na-phosphate buffer, pH 7.4) was used. Theresulting conjugate was purified by ultrafiltration (10 kDa, Amicon,Millipore) with deionized water. The protein concentration wasdetermined by bicinchoninic acid (BCA) assay (Pierce).

Successful conjugation was confirmed by SDS-PAGE as shown in FIG. 4 a.Marker M is PageRuler Plus Prestained Protein Ladder (ThermoScientific). Conjugate samples are shifted towards higher massescompared with unconjugated CRM₁₉₇.

The oligosaccharide/CRM₁₉₇ ratio was determined by MALDI-TOF MS. Themass analysis of CRM₁₉₇ yielded a m/z ion at 58.2 kDa. The mass analysisof the conjugate yielded a major m/z ion at 67.7 kDa and further peaks˜1000 Da apart, corresponding to conjugates of different valencies (FIG.4 b). An average of 9.6 PS-I pentasaccharide 1 molecules were loaded onone CRM₁₉₇ protein, resulting in conjugate 1b.

Knowing the protein concentration of the conjugate, as determined bybicinchoninic acid (BCA) assay, and the average sugar loading, thecarbohydrate content was calculated to 300±46 μg/mL (mean±SD) andverified by colorimetric anthrone assay (302±76 μg/mL), an approvedmethod for the carbohydrate determination of the licensed pneumococcalconjugate vaccine Prevenar (Pfizer).

SDS-PAGE

Pentasaccharide 1-CRM₁₉₇ conjugate and unconjugated CRM₁₉₇ weredissolved in Lämmli buffer (0.125 M Tris, 20% (v/v) glycerol, 4% (w/v)SDS, 5% (v/v) beta-mercaptoethanol, bromophenol, pH 6.8) and boiled at95° C. for 5 minutes. Samples were run in 10% polyacrylamide gels andstained with 0.025% (w/v) Coomassie Brilliant blue R-250 in an aqueoussolution containing 40% (v/v) methanol and 7% (v/v) acetic acid.

MALDI-TOF Mass Spectrometry

Conjugation was confirmed by matrix-assisted laserdesorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)using an Autoflex™ Speed instrument (Bruker Daltonics, Bremen, Germany).The mass spectrometer was operated in positive linear mode. Spectra wereacquired over an m/z range from 50,000 to 85,000 Da and data wasanalyzed with the FlexAnalysis software provided with the instrument.2′,4′-dihydroxyacetonephenone (DHAP) was used as matrix, samples werespotted using the dried droplet technique.

Anthrone Assay

Anthrone assays were performed in 96-well format in a modified assayaccording to Leyva et al., Biologicals 36:134-141, 2008. Briefly, 75 μLof anthrone reagent (0.1% (w/v) in concentrated sulfuric acid) was addedto each well of a 96-well microtiter plate containing 25 μL of standardsolutions, sample dilutions and blank. Plates were first placed at 4° C.for 10 minutes, then incubated at 100° C. for 20 minutes, and cooleddown at room temperature for 20 minutes. Absorbance at 579 nm wasdetermined in a microplate reader. Colorimetric response was compared toa standard curve based on glucose and rhamnose in a 3:2 molar ratio.

EXAMPLE 4 Immunization and Monoclonal Antibodies

To test the immunogenicity of the PS-I pentasaccharide hapten, threegroups of six female C57BL/6 mice each were immunized subcutaneously(s.c.) with conjugate (one group without adjuvant, one group withFreund's adjuvant, one group with Alum adjuvant). Each mouse received anamount of conjugate corresponding to 3 μg PS-I pentasaccharide 1antigen. Initial immunizations (priming) was followed by an immunizationafter two weeks (boosting). Sera were collected in one-week intervals.IgG antibody responses were evaluated by microarray. PS-Ipentasaccharide 1 in three different concentrations (1, 0.5 and 0.1 mM),CRM₁₉₇ (1, 0.5 and 0.1 μM) and bovine serum albumin (BSA)-spacer-GlcNAcconjugate (1, 0.5 and 0.1 μM) were spotted in triplicate onto thesurface of the microarray slides (N-hydroxysuccinimid ester-activatedglass slides (CodeLink)) as shown in FIG. 5. BSA-spacer-GlcNAc was usedto assess immunogenicity against the spacer moiety of the conjugate. Asnegative controls, phosphate-buffer saline (PBS), as well as twounrelated oligosaccharides (both at a concentration of 1 mM) were alsoincluded. Microarrays were designed such that high-throughput analysisof 64 samples per array was possible.

PS-I pentasaccharide-specific IgG antibody responses were identified inpooled sera of three groups (each n=6) of immunized mice after priming,and more pronounced after boosting (week 3), as determined by microarrayanalysis (FIG. 6).

IgG antibody responses were quantified by determination of thefluorescence intensity values using the sera of individual mice. Whilethe conjugate already showed immunogenicity without adjuvant (FIG. 7,left diagram, white bars), IgG titers against PS-I pentasaccharide weremarkedly increased when Freund's adjuvant was used (light grey bars),and, more pronounced, with Alum adjuvant (dark grey bars). IgG antibodytiters against the carrier protein CRM₁₉₇ were lower in mice immunizedwithout adjuvant than in mice immunized with Freund's and Alum adjuvant(FIG. 7, central diagram). There was no IgG response against the spacermoiety in mice immunized without adjuvant, but in mice immunized withFreund's and Alum adjuvants (FIG. 7, right diagram).

As an IgG-specific detection antibody, Anti-Mouse IgG (wholemolecule)-FITC (Sigma) was used in the tests of FIGS. 6 and 7. Slideswere analyzed on a GenePix Pro 4300A microarray scanner and data wasanalyzed using the GenePix Pro 7 software (both Molecular Devices).Individual mice sera at week 0 (‘preblee’), week 2 (‘prime’) and week 3(‘boosted’) were analyzed by microarray (FIG. 6). Total fluorescenceintensity values were determined with the GenePix Pro 7 software andbackground fluorescence (PBS) was subtracted for each value. Data shownis mean±S.E.M. (standard error of the mean) for n=6 values. “Unrel. OS”in FIG. 5 means unrelated oligosaccharide.

To get an insight into the subclasses of IgG antibodies raised againstPS-I pentasaccharide, microarray analysis with pooled sera usingsubclass-specific detection antibodies against IgG1, IgG2a and IgG3 wasperformed.

FIG. 8 shows the isotype analysis of the IgG immune response bymicroarray. Pooled sera at a 1:100 dilution were analyzed withisotype-specific detection antibodies (anti-IgG1, Invitrogen A21125;anti-IgG2a, Invitrogen A21241; anti-IgG3, Invitrogen A21151). Data shownis mean, n=6, S.E.M., normalized to background fluorescence intensity,of mice after boosting (week 3).

As evident from FIG. 8, while antibodies against PS-I are almostexclusively of the IgG1 subtype in mice immunized with conjugate withoutadjuvant (left panel) or Alum adjuvant (right panel), mice immunizedwith Freund's adjuvant show a relatively high proportion of antibodiesof the IgG2a and IgG3 subclasses in addition to IgG1. IgG3 and IgG2a aremainly induced by T-cell independent antigens such as polysaccharides,while IgG1 is mainly T-cell dependent and directed against proteinantigens.

To assess whether antibodies raised with PS-I pentasaccharide antigen 1recognize substructures of the antigen as well, which allows to definethe minimal epitope, microarray slides with substructures 35-39 inaddition to 1 were prepared (FIG. 9). CRM₁₉₇, BSA-spacer-GlcNAc wereincluded as well as two unrelated oligosaccharides and PBS as negativecontrols. This array was used to assess immune responses in pooled seraof the three groups of mice immunized with conjugate.

FIG. 10 shows the deletion sequence analysis of the immune response ofmice immunized with glycoconjugate without adjuvant. Pooled sera of mice(n=6) were analyzed on deletion sequence microarray as in FIG. 9, usingAlexa Fluor 635 goat anti-mouse IgG (Invitrogen) as detection antibody.Unrel. OS, unrelated oligosaccharide.

FIG. 11 shows the deletion sequence analysis of the immune response ofmice immunized with glycoconjugate and Freund's adjuvant.

FIG. 11 shows the deletion sequence analysis of the immune response ofmice immunized with glycoconjugate and Alum adjuvant.

As shown in FIGS. 10 and 11, sera of mice immunized without adjuvant orwith Freund's adjuvant contain antibodies against substructure withrhamnose, while the IgG responses against disaccharide 38 is generallyhigher than those against trisaccharide 37, albeit 37 is closer to theoriginal PS-I pentasaccharide antigen 1 used for immunization. The IgGantibody response in mice immunized with Alum adjuvant shows a morespecific reactivity against the PS-I pentasaccharide with lower titersagainst deletion sequences 38 and 37 (FIG. 12). No antibody responseagainst oligoglucose disaccharide 35 nor trisaccharide 36 was detectedin any of the groups. Disaccharide 38 may be the minimal epitope of thePS-I pentasaccharide.

Monoclonal antibodies were generated with the traditional hybridomatechnique [Köhler and Milstein, 1975]. Three monoclonal antibodies(mAbs), 2C5, 10A1 and 10D6, were selected for evaluation with deletionsequence microarray and isotype-specific detection antibodies. All threemABs showed identical patterns on the microarray, exclusively bound toPS-I pentasaccharide 1 but none of the substructures, and were of theIgG1 subtype (FIG. 13).

FIG. 13 shows different monoclonal antibodies against PS-I. One mouse ofthe Alum group was subjected to a second boosting immunization (s.c.) atweek 5 and three final boostings (intraperitoneal, i.p.) at threeconsecutive days in week 7. One day after final boosting, the mouse wassacrificed, the spleen was removed and subjected to monoclonal antibodydevelopment. After three rounds of subcloning, supernatants of threemonoclonal antibodies (mAB)-producing clones, 2C5, 10A1 and 10D6, weresubjected to isotype analysis as in FIG. 8, using hybridoma supernatantsin a 1:3125 dilution.

Immunizations

Six to eight-weeks old female C57BL/6 mice were immunized s.c. withconjugate corresponding to 3 μg PS-I pentasaccharide 1 with Freund's(priming immunizations with Freund's Complete Adjuvant, boostingimmunizations with Freund's Incomplete Adjuvant, both Sigma) orAluminium Hydroxide Gel Adjuvant (Brenntag Biosector, Frederikssund,Denmark), or without adjuvant. Mice received boosting injections after 2weeks. For all immunizations, antigen was diluted in sterile PBS to atotal injection volume of 100 μL per mouse. Blood was collected inone-week intervals via the tail vein and erythrocytes separated fromserum by centrifugation. Serum serum antibody responses were analyzed bymicroarray. One mouse of the Alum group received a second boostinginjection s.c. at week 5 after first immunization, and, prior to beingsacrificed, three final boosting injections via the intraperitoneal(i.p.) route, on three consecutive days at week 7.

Preparation of Microarrays

Oligosaccharides bearing an amine linker, or proteins, were dissolved insodium phosphate buffer (50 mM, pH 8.5) and printed robotically using apiezoelectric spotting device (S11, Scienion, Berlin, Germany) ontoNHS-activated glass slides (CodeLink). Slides were incubated in a humidchamber to complete reaction for 24 hours and stored in an anhydrousenvironment. Prior to the experiment, remaining succinimidyl groups werequenched by incubating slides in 100 mM ethanolamine in sodium phosphatebuffer (pH 9, 50 mM) for 1 hour at 50° C. Slides were rinsed three timeswith deionized water and dried by centrifugation.

Microarray Binding Assays

The quenched array slides were blocked for 1 hour with 1% (w/v) BSA inPBS, then washed three times with PBS and dried by centrifugation. AFlexWell 64 (Grace Bio-Labs, Bend, Oreg., USA) grid was applied to theslides. Resulting 64 wells were used for 64 individual experiments.Slides were incubated with sera dilutions or hybridoma supernatants (alldilutions were prepared with PBS) for 1 hour at room temperature in ahumid chamber, washed three times with PBS-Tween-20 (0.1% v/v) and driedby centrifugation. Then, slides were incubated with fluorescence-labeleddetection antibody diluted in 1% BSA in PBS (w/v) for 1 hour at roomtemperature in a humid chamber. Slides were washed three times withPBS-Tween-20 (0.1% v/v) and rinsed once with deionized water and driedby centrifugation. Slides were scanned with a GenePix 4300A scanner(Molecular Devices) using the GenePix Pro 7 software. Detectionantibodies used were Anti-Mouse IgG (whole molecule)-FITC (Sigma), AlexaFluor 635 Goat Anti-Mouse IgG (H+L) (Life Technologies) and Alexa Fluor594 Goat Anti-Mouse IgG1 (γ1) (Life Technologies) in 1:400 dilutions, aswell as Alexa Fluor 647 Goat Anti-Mouse IgG2a (γ2a) and Alexa Fluor 488Goat Anti-Mouse IgG3 (γ3) (Life Technologies) in 1:200 dilutions.

Monoclonal Antibodies

Monoclonal antibodes (mABs) were generated using the standard method byKöhler and Milstein, 1975. Briefly, spleenocytes of one mouse were fusedwith 10⁸ mouse myeloma cells in the presence of 50% PEG 1500. Fusedcells were selected with complete growth medium (IMDM supplemented with10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 24 μMbeta-mercaptoethanol, 100 μM hypoxanthine, 16 μM thymidine,non-essential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin,50 μg/mL gentamycin, 10% hybridoma cloning supplement (BM Condimed H1,Roche)) with 0.4 μM aminopterin. Cells were maintained at 37° C. at 5%CO₂. Hybridoma cells were subjected to three consecutive subcloningsteps by limited dilution. Clones producing antibodies against PS-Ipentasaccharide were identified by microarray analysis.

The invention claimed is:
 1. A method for preparing a pentasaccharidehaving the sequenceα-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcpand represented by structure 1 or 1′

or a derivative thereof in which at least one hydroxyl group thereof issubstituted by at least one other functional group or atom, said methodcomprising a) assembling a monosaccharide building block 2′, wherein thespecific protected amino linker of building block 2 is replaced by adifferent protected or unprotected linker L, and building blocks 3 or 4to yield the corresponding disaccharide 21′, reacting the disaccharide21′ with building block 4 to form the trisaccharide 23′, subjecting thetrisaccharide 23′ to a bis-glycosylation reaction with 2 molecules ofbuilding block 5 to yield the corresponding fully protectedpentasaccharide 24′ and finally, after deprotection, to yieldpentasaccharide 1′, wherein the specific amino linker of pentasaccharide1 is replaced by a different linker L, or b) assembling a monosaccharidebuilding block 2′, wherein the specific protected amino linker ofbuilding block 2 is replaced by a different protected or unprotectedlinker L, and building block 27 to yield the corresponding disaccharide30′, reacting the disaccharide 30′ with building block 4 or 29 to formthe corresponding protected trisaccharide 32′, deprotecting thetrisaccharide 32′ to obtain trisaccharide 33′, subjecting thetrisaccharide 33′ to a bis-glycosylation reaction with 2 molecules ofbuilding block 5 to yield the corresponding fully protectedpentasaccharide 34′ and finally, after deprotection, to yieldpentasaccharide 1′, wherein the specific amino linker of pentasaccharide1 is replaced by a different linker L, wherein the different linker L is(CH₂)_(n)NH₂, with n being an integer from 2 to 50, and where 2, 2′, 3,4, 5, 21′, 23′, 24′, 27, 29, 30′, 32′, 33′, 34′ are defined as follows:


2. The method according to claim 1, wherein the different linker L ofpentasaccharide 1′ is (CH₂)_(n)NH₂, with n being an integer from 3 to10.